DNA damage and repair in human leukocytes exposed to styrene-7,8-oxide measured by the comet assay

Toxicology Letters 126 (2002) 61 – 68 www.elsevier.com/locate/toxlet

DNA damage and repair in human leukocytes exposed to styrene-7,8-oxide measured by the comet assay Blanca Laffon a,b, Eduardo Pa´saro b, Josefina Me´ndez a,* a

Dept. de Biologia Celular y Molecular, Facultad de Ciencias, Uni6ersidade da Corun˜a, Campus A Zapateira s/n, 15071 La Corun˜a, Spain b Instituto de Ciencias de la Salud, Uni6ersidade da Corun˜a, La Corun˜a, Spain Received 25 May 2001; received in revised form 20 August 2001; accepted 29 August 2001

Abstract Styrene-7,8-oxide (SO) is produced by cytochrome p450 monooxygenases as the main mammalian metabolite of styrene, an important industrial chemical present at high concentrations in the ambient air of fiberglass-reinforced plastic plants. Previous studies have shown positive results for SO in the induction of several cytogenetic endpoints in vitro. In this work we have evaluated, by means of the comet assay, the potential of SO to act as a DNA damaging agent in human peripheral leukocytes and the ability of white blood cells to repair the DNA damage induced by this compound. Our results show that SO induces DNA damage at concentrations higher than 50 mM in a dose-dependent manner, and that the lesions produced by SO are efficiently removed within a few hours after the end of treatment. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Styrene-7,8-oxide; Comet assay; Human leukocytes; DNA repair

1. Introduction Styrene-7,8-oxide (SO) is the primary mammalian metabolite of styrene, an organic solvent widely used in the production of plastics, resins, insulators and synthetic rubber. The highest human exposure to styrene takes place in fiberglassreinforced plastic plants, where the compound is inhaled during by-hand-lamination procedures (Miller et al., 1994). In such factories, SO is produced from airborne styrene in the presence of * Corresponding author. Tel.: + 34-981-167000; fax: +34981-167065. E-mail address: [email protected] (J. Me´ndez).

air and light and also when peroxides are added to the resin as polymerisation-reaction initiators. The concentration of SO in the air at the work place has been calculated to be 1/1000 of the concurrent air concentration of styrene (Pfa¨ffli and Sa¨a¨ma¨nen, 1993). In vivo, styrene is metabolised to SO by cytochrome P450 monooxygenases, the isoforms CYP2B6 and CYP2E1 (present in human liver and lung) and CYP2F1 (present in human lung) being the most effective in the transformation (Nakajima et al., 1994; Kim et al., 1997). Previous studies performed in several mammalian cell systems and lower eukaryotes treated in vitro with

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SO have demonstrated the induction of sisterchromatid exchanges, micronuclei and chromosomal aberrations (reviewed in Barale, 1991; Scott and Preston, 1994). Also, changes in the expression of some genes involved in cell cycle and apoptosis regulation have been described in human lymphocytes exposed to SO (Laffon et al., 2001). None of the remainder styrene metabolites have shown genotoxic activity (Barale, 1991). Moreover, SO is thought to be a potential carcinogenic hazard in occupationally exposed workers, especially in terms of lymphatic and hematopoietic malignancies (Wong, 1990; Kolstad et al., 1994; Newhook et al., 1994). On the basis of the published studies, the International Agency for Research on Cancer has found enough evidence of SO carcinogenicity in animals and has classified this compound as probably carcinogenic in humans (Group 2A) (IARC, 1994). The alkaline single cell gel electrophoresis (comet) assay is a rapid, simple and sensitive genotoxicity test for the measurement of radiation or chemically induced DNA damage and repair in viable cells (reviewed in McKelvey-Martin et al., 1993; Fairbairn et al., 1995; Rojas et al., 1999). The cells are embedded in agarose gel on microscope slides, lysed and subjected to electrophoresis. Intact DNA, with very high molecular weight, remains in place, but damaged DNA, denatured in the alkaline buffer, migrates into the gel. After staining with a fluorescent dye, the cell takes the appearance of a ‘comet’, with the head containing unbroken DNA and the tail, streaming away in the direction of electrophoresis, containing broken DNA. Vodicka et al. (1995) used comet assay methodology to evaluate genotoxic risk associated to styrene exposure, and found significantly higher levels of DNA damage among the group of lamination workers as compared to a control population. The objective of the present work was to evaluate, by means of the comet assay, the potential of SO to act as a DNA damaging agent, in addition to its already-described cytogenetic damaging properties, in human peripheral leukocytes. Moreover, we aim to determine the ability of white blood cells to repair the DNA damage induced by this compound.

2. Materials and methods

2.1. Chemicals and subjects SO (CAS No. 96-09-3) and dimethylsulfoxide (DMSO) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). SO solutions were prepared in DMSO and were added to the culture media to obtain final concentrations of 10, 20, 50, 100 and 200 mM. DMSO was used as the solvent control. In the repair study only 50 and 200 mM SO treatments and solvent control were used. The maximum concentration of the test substance was determined as the concentration at which cell viability was above 75%, thus avoiding false positive responses due to cytotoxicity, as indicated by Henderson et al. (1998). Heparinised venous blood from four healthy donors aged 23–30, two female (donors 1 and 3) and two male (donors 2 and 4), was obtained by venipunction. These subjects were non-smokers and had not been exposed to any specific chemical substance or radiation during the previous 2 months.

2.2. Leukocyte isolation and SO treatment Mononuclear leukocytes were isolated by means of a Ficoll density gradient. Whole blood was diluted (1:1) in phosphate buffer solution (PBS) pH 7.4 and centrifuged over half a volume of lymphocyte isolation medium (Rafer, Zaragoza, Spain) at 2100 rpm for 30 min. The buffy coat was removed and washed twice in PBS at 1500 rpm for 10 min. Leukocytes were then suspended in RPMI 1640 medium containing the SO treatments or DMSO to obtain 5×105 cells/ml, and were maintained at 37 °C for 30 min. Cells were collected by centrifugation at 9000 rpm for 3 min and washed in PBS. In the case of the repair study, cells were then suspended in fresh RPMI 1640 medium and incubated again at 37 °C for 30, 120 or 240 min. Cell viability was estimated by trypan blue exclusion and was higher than 75% in all cases.

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2.3. Comet assay The alkaline comet assay was performed essentially as described by Singh et al. (1988). Briefly, 2.5 ×105 cells were mixed with 80 ml of low-melting-point agarose (LMA) (Gibco BRL, Paisley, Scotland) at 0.7% in PBS and added to frosted microscope slides previously coated with a 130 ml layer of 1% normal-melting-point agarose (Gibco BRL, Paisley, Scotland) in PBS. Slides were placed on ice for 10 min and a third layer of 80 ml of LMA was applied and allowed to solidify again on ice. Slides were then carefully immersed in lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, pH 10, with 1% Triton X-100 added just before use), and kept at 4 °C for 1 h. All of the steps after lysis were conducted under dim light to prevent the occurrence of additional DNA damage. Slides were placed in a horizontal gel electrophoresis tank with alkaline solution (1 mM Na2EDTA, 300 mM NaOH, pH \ 13) into an ice bath, and they were left for 20 min to allow DNA unwinding and alkali-labile site expression. Electrophoresis was carried out for 20 min at 0.83 V/cm. After electrophoresis, slides were rinsed three times for 5 min each with neutralising solution (0.4 M Tris–HCl, pH 7.5), stained with 60 ml of 5 mg/ml 4,6-diamidino-2-phenylindole (DAPI) in antifade solution, and covered with a coverslip. The preparations were kept in a humidified sealed box to prevent drying of the gel and analysed within 24 h to avoid excessive diffusion of the DNA in the gel. Two slides were prepared for each experimental concentration and donor.

2.4. Imaging and analysis All slides were coded before they were scored. They were examined at 400× magnification in a Leica DM-RXA fluorescence microscope equipped with a 100 W mercury lamp. Images of 100 randomly selected cells (50 per replicate slide) were analysed from each sample. As recommended by Hartmann and Speit (1997), DNA clouds were excluded from the evaluation to avoid confusion due to cytotoxicity, since these are assumed to represent dead cells. Image capture and analyses were performed by use of the QWIN

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Comet software (Leica Imaging Systems, Cambridge, UK). This software, based on densitometric measures, divides the cell in head and tail by assuming head shape as circular, and considering everything outside the head circumference and towards electrophoresis direction as comet tail. Tail length, measured from the estimated center of the cell, percentage of DNA in the comet tail (%TDNA) and tail moment, which is calculated by multiplying tail length by %TDNA, were measured as DNA damage parameters.

2.5. Statistical e6aluation Since distribution of tail length, tail moment and %TDNA departed significantly from normality (Kolmogorov-Smirnov goodness of fit test), differences between control cells and those exposed to different SO concentrations were evaluated by application of the non-parametric Mann –Whitney U-test. The associations between two variables were analysed by Pearson’s correlation. The level of significance was at 0.01, but 0.05 was also taken into account when the difference between two groups was considerable but it did not reach the level of significance of 0.01. All analyses were conducted using the SPSS for Windows statistical package, version 10.0 (Illinois, USA).

3. Results and discussion DNA single-strand breaks or alkali-labile sites are induced by a great variety of genotoxic substances. By means of the alkaline version of the comet assay, single-strand breaks or adducts that drive to AP (apurinic or apirimidinic) alkali-labile site formation can be detected non-specifically (Singh et al., 1988; Olive et al., 1992). This methodology presents the advantage that DNA damage can be evaluated at the level of single cells, in comparison with other methods to analyse DNA damage (alkaline elution, alkaline unwinding, etc.). Table 1 shows the mean values of tail length, %TDNA and tail moment in leukocytes from each donor exposed to increasing doses of SO or

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DMSO as control, and Fig. 1 shows averaged values of these three parameters based on pooled data from the four donors. In general, significant increases for all the three parameters evaluated have been observed starting at a concentration of 100 mM SO. Tail length values are less dispersed than the other ones, interindividually and intraindividually, as reflected by the smaller mean standard errors in proportion to the mean values. These results agree with those presented by Bastlova´ et al. (1995), which describe tail length increments in human leukocytes treated with 50 and 100 mM SO, and by Dypbukt et al., 1992, which show single-strand breaks (evaluated by alkaline elution) in Pc12 cells exposed to 30 and 100 mM SO. The three evaluated variables are highly correlated with SO concentrations (r= 0.978, P 5 0.01 for tail length; r = 0.915, P 5 0.05 for %TDNA and r =0.885, P 50.05 for tail moment). Thus a good dose– response relationship is observed, at least at the doses employed. DNA damage induced by SO is thought to be mediated by alkylation of DNA bases, preferentially at the N7 position of guanine (Vodicka and Hemminki, 1988; Prakash and Gibson, 1992).

Alkylation of the imidazole ring nitrogens in nucleotides produced by SO and many other epoxides can lead to instability of the DNA structure and, as a result, to imidazole ring opening and formation of AP sites (Chovanec et al., 1998). These alkali-labile AP sites are converted to strand breaks during the alkaline unwinding period of the comet assay. In addition, DNA damage detected after SO exposure may be caused by oxidative stress, as indicated by Marczynski et al. (1997), who described high molecular weight DNA fragmentation following human blood exposure to SO, and suggested this may be due to disruption of pre-existing oxidative status and to alteration of the balance between oxidants and antioxidants in blood cells. There is no real consensus on the most correct measure for the evaluation of DNA damage in the comet assay. Tail length indicates the extent of migration of the genetic material in the direction of the anode (Singh et al., 1988) and is expected to be proportional to the level of single-strand breaks and alkali-labile sites. However, the criteria to identify the trailing and leading edge of the migrating DNA is not unanimous but is depen-

Table 1 Mean values of tail length (TL, mm), percentage of DNA in the comet tail (%TDNA) and tail moment (TM, mm) in human leukocytes exposed to SO Control

10 mM

Donor 1 TL %TDNA TM

56.56 0.654 51.37

48.75 0.116 6.54

58.43a 0.411 33.42

Donor 2 TL %TDNA TM

54.03 1.117 118.24

54.03 0.120 8.28

63.24a 1.435a 139.65a

Donor 3 TL %TDNA TM

41.35 0.211 17.36

44.71a 0.274a 25.04a

40.07 0.031 1.76

Donor 4 TL %TDNA TM

47.83 0.316 21.60

46.73 0.311 22.33

41.58 0.077 3.96

a

20 mM

50 mM

100 mM

200 mM

48.47 0.030 4.85

79.64a 2.240a 230.47a

75.57a 1.370a 110.99a

72.21a 0.120 149.04a

82.86a 3.144a 302.15a

95.51a 4.542a 444.15a

42.96 0.212a 14.25

45.13a 0.226a 15.55

60.03a 0.744a 49.55a

46.78 0.228 17.91

49.35a 0.225 14.23

58.39a 0.719a 50.98a

P50.01, significant difference with regard to the corresponding control for each donor.

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dent on the researcher and/or image analysis software used. Measurement of the percentage of migrated DNA has been shown to be proportional to the frequency of DNA damage (Olive et al., 1990). Nevertheless, this parameter assumes signal linearity in quantifying the amount of DNA, ranging over multiple orders of magnitude between head and tail, and also assumes that the efficiency of the fluorescent dye is equal in the staining of migrated and non-migrated DNA (Tice et al., 2000). Tail length increases with dose at low levels of damage, but this soon stops, and then the increase is in %TDNA, i.e. once the tail is established its length is constant and tail intensity rather than length increases. The more likely explanation is based in the migration of DNA loops. One break in a loop relaxes supercoiling and allows the DNA to extend into the tail, being tail length presumably determined by the length of the loops (Collins et al., 1997). As for tail moment, although its concept is clear as introduced by Olive et al. (1990) (the result of multiplying a measure of tail length by a measure of DNA amount in the comet tail), no agreement has been reached among investigators as to the most appropriate manner in which to calculate it. It has been suggested that the main advantage of tail moment is that the amount of migrated DNA and migration distance are represented in a single value (Bo¨ cker et al., 1997). However, comet tails with different lengths and relative amounts of DNA may have the same tail moment (Hellman et al., 1995). On the other hand, Tice (1995) stated that some agents (e.g. ionising radiation) induce long, thin tails while others (e.g. cyclophosphamide) induce short, thick tails. Moreover, Yendle et al. (1997) indicated that there are two types of comet tail with different appearances (stellate versus diffused), consistent with the existence of two mechanistically distinct ways of producing comet tails. Therefore, it seems reasonable that no single parameter is valid to analyse DNA damage induced by all compounds. On the basis

Fig. 1.

Fig. 1. Comet tail length (A), %TDNA (B) and tail moment (C) induced by SO in human leukocytes: averaged values based on pooled data from the four donors. Bars represent mean standard error.

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of our results, which show less dispersed values and a better dose– response relationship for tail length, we think that this parameter is the most adequate to evaluate DNA damage induced by SO. In the repair study only tail length has been measured as DNA-damage parameter. Table 2 displays tail length values in leukocytes, from every donor, exposed to SO or DMSO for 30 min and varying DNA-repair times, and Fig. 2 shows the results of the repair study based on pooled data from the four donors. Results of this study reveal that the repair of DNA damage induced by SO takes place efficiently but in a relatively slow manner. In fact, after 30 min of incubation in fresh medium, the decrease of the difference in tail length between control cells and those treated with SO is noteworthy, although the significance is maintained in all the treated leukocytes for donor 1 and in 200 mM SO-treated cells for donors 2, 3 and 4. After 120 min, only leukocytes exposed to SO 200 mM from donors 1, 2 and 3 show a slight but significant increase in DNA Table 2 Mean comet tail length (mm) in human leukocytes exposed to SO for 30 min and then to fresh medium for 0, 30, 120 and 240 min to allow repair of DNA damage Treatment

0 min

30 min

120 min

240 min

Donor 1 Control 50 mm SO 200 mm SO

56.56 48.47 75.57b

41.79 49.61b 56.59b

47.67 45.64 52.71b

46.63 46.91 47.06

Donor 2 Control 50 mm SO 200 mm SO

54.03 72.21 95.51b

43.99 42.28 51.71b

49.90 49.60 52.81a

45.95 43.25 45.99

Donor 3 Control 50 mm SO 200 mm SO

41.35 42.96 60.03b

47.51 47.09 58.57b

45.74 45.02 50.80b

47.78 46.58 53.15b

Donor 4 Control 50 mm SO 200 mm SO

47.83 46.78 58.39b

44.04 46.82 67.10b

51.58 44.34 49.81

53.18 51.07 54.30

a

P50.05. P50.01, significant difference with regard to the corresponding control for each time point. b

Fig. 2. Comet tail length in human leukocytes treated with SO for 30 min and then with fresh medium for 0, 30, 120 and 240 min: averaged values based on pooled data from the four donors. Bars represent mean standard error.

damage, and after 240 min comet tail length returns to control values in all the cells, except for donor 3 leukocytes exposed to the higher SO dose. In the aforementioned studies of Bastlova´ et al. (1995), Dypbukt et al. (1992) the SO treatment time is longer than that in our study (1 h vs. 30 min), but removal of DNA damage is also evident at 2 and 3 h and is complete after 24 h for the highest concentrations, although intermediate times are not evaluated. Schmezer et al. (2001) established 15 min as the optimal time to analyse the repair of DNA damage induced by exposure to the clastogen bleomycin in peripheral lymphocytes, but these cells were stimulated with phytohemagglutinin for 20 h before the treatment, causing cells to pass through resting G0 phase into active G1 phase of the cell cycle. The delay in the repair process in non-stimulated cells is due to their scarcity in DNA precursors (dNTPs), that determine the process to take place in a limited rate (McKelvey-Martin et al., 1993; Collins et al., 1997). On the other hand, repair of AP alkalilabile sites, induced by alkylating agents like SO, occurs more slowly than the repair of DNA single-strand breaks, and alkylated sites are efficiently converted into single-strand breaks during incubation time, contributing to the relatively slow rejoining kinetics (Chovanec et al., 1998).

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In summary, our results show that SO induces DNA damage in human peripheral blood leukocytes exposed in vitro to concentrations higher than 50 mM in a dose-dependent manner, and that the lesions produced by SO are efficiently removed within a few hours after the end of treatment.

Acknowledgements This investigation has been supported by a fellowship from the University of A Corun˜ a (to B. Laffon) and by a grant from the Xunta de Galicia (XUGA 10605B98).

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