Urinary N3 adenine DNA adducts in humans occupationally exposed to styrene

Toxicology Letters 197 (2010) 183–187

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Urinary N3 adenine DNA adducts in humans occupationally exposed to styrene Petr Mikeˇs a , Marek Koˇrínek a , Igor Linhart b,∗ , Jan Krouˇzelka b , Ludmila Dabrowská c , Vladimír Stránsky´ c , Jaroslav Mráz c a

Apigenex, Podˇebradská 186, CZ-180 66 Prague, Czech Republic Institute of Chemical Technology, Prague, Faculty of Chemical Technology, Department of Organic Chemistry, Technická 5, CZ-166 28 Prague, Czech Republic c National Institute of Public Health, Sˇ robárova 48, CZ-100 42 Prague, Czech Republic b

a r t i c l e

i n f o

Article history: Received 7 April 2010 Received in revised form 19 May 2010 Accepted 21 May 2010 Available online 27 May 2010 Keywords: Styrene Occupational exposure Urinary DNA adducts Biomarkers 3-Alkyladenines Biomarkers of effective dose

a b s t r a c t Urine samples from humans occupationally exposed to styrene, with mandelic acid levels ranging from 400 to 1145 mg/g creatinine and from 68 to 400 mg/g creatinine for high and low exposure group, respectively, were analysed for N3 adenine DNA adducts, namely, 3-(2-hydroxy-1-phenylethyl)adenine (N3␣A) and 3-(2-hydroxy-2-phenylethyl)adenine (N3␤A). A sensitive LC-ESI–MSMS method was developed with the limit of quantification of 1 pg/mL for both analytes. Peaks corresponding to N3␣A and/or N3␤A were found in seven of nine end-of-shift samples of the high exposure group and in six of 19 end-of-shift samples of the low exposure group. Concentration of N3␣A + N3␤A amounted to 2.8 ± 1.6 pg/mL (mean ± S.D.; n = 9) and 1.8 ± 1.3 pg/mL (mean ± S.D.; n = 19) in the high and low exposure group, respectively. Of other 10 samples taken the next morning after exposure, two contained low but quantifiable concentrations of N3␣A and none contained N3␤A. However, interfering peaks were detected also in some control urine samples. Out of 22 controls, six and two samples contained peaks co-eluting with N3␣A and N3␤A, respectively. Therefore, the method used was found insufficiently specific to be applicable for biological monitoring. Comparing the excretion of N3␣A + N3␤A to that reported previously in mice it can be estimated that at the same absorbed dose, humans excreted not more than 1/30 of the amount of adenine adducts excreted by mice. As a consequence, the damage to DNA caused by styrene 7,8-oxide (SO), a reactive metabolite of styrene, appears to be much lower in humans than in mice. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Styrene, an industrial monomer produced on a mass scale, is widely used for production of polymers, resins, reinforced plastics and latex paints (Miller et al., 1994; IARC, 1994). It has been demonstrated by numerous experimental studies in vitro and in vivo that styrene, after metabolic activation to styrene 7,8-oxide (SO), is capable of binding to the DNA (for reviews see Phillips and Farmer, 1994; Hemminki et al., 2000). DNA adduction may then lead to the cytogenetic damage (Mäki-Paakkonen et al., 1991; Walles et al., 1993) and in the worst case also to carcinogenicity (IARC, 2002). SO has been classified as a probable human carcinogen, group 2A (IARC, 1994) while styrene itself as a possible human carcinogen, group 2B (IARC, 2002). The possible carcinogenicity of styrene is believed to be related to the binding of SO to DNA which occurs at multiple sites (Koskinen et al., 2001; Vodicka et al., 1999, 2002). Several studies report on the specific DNA adducts in white blood cells from workers occupationally exposed to styrene. These include O6 - and N2 -substituted guanine adducts (Vodicka et al., 1993, 1994, 1999;

∗ Corresponding author. Tel.: +420 220 444 165; fax: +420 220 444 288. E-mail address: [email protected] (I. Linhart). 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.05.015

Horvath et al., 1994) and, more recently, N1-substituted adenine adducts (Koskinen et al., 2001a, Vodicka et al., 2003). In contrast, when DNA was reacted with SO in vitro the main sites of attack were invariably N7 at guanine and N3 at adenine representing about 97% of the total DNA alkylation (Koskinen et al., 2000). These positions represent major binding sites also for some other alkylating agents (Shuker and Farmer, 1992; Koskinen and Plna, 2000). However, neither N7 guanine nor N3 adenine adducts were found in the lymphocytes of workers occupationally exposed to styrene. Alkylation at these sites results in a positively charged imidazole ring of the purine base making the N-glycosidic bond more labile and prone to hydrolysis, so that both N7 guanine and N3 adenine adducts are rapidly cleaved off the DNA molecule (Mueller and Risenbrand, 1985; Margison et al., 1973; Fujii et al., 1980). Cleavage of the N3 adenine adducts derived from SO is shown in Fig. 1. The easily depurinating adducts are then excreted as 7-alkylguanines and 3-alkyladenines in urine and may therefore serve as useful non-invasive biomarkers of both exposure and response to mutagenic and carcinogenic agents (Shuker and Farmer, 1992; Timbrell, 1998). Recent development in the analytical techniques based on mass spectrometry made possible to detect and quantify nucleobase DNA adducts in the urine at concentrations which are relevant

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P. Mikeˇs et al. / Toxicology Letters 197 (2010) 183–187 2.2. Authentic standards Authentic standards of 3-(2-hydroxy-1-phenylethyl)adenine (N3␣A) and 3(2-hydroxy-2-phenylethyl)adenine (N3␤A) were prepared in our laboratory as described by Krouˇzelka et al. (2008). These standards were used for validation of linearity of the calibration curve as well as for the adduct quantification using a standard addition method. 2.3. Sample preparation Aliquots (1.8 mL) of human urine were mixed with 200 ␮L of 66% trichloroacetic acid solution and vortexed for 10 min. The samples were centrifuged (1300 rpm, 10 min) and 1.5 mL of the supernatants were then transferred onto OasisTM MCX 60 mg SPE columns (strong cation exchange and reversed-phase sorbent, Waters), which were preconditioned with 2 mL of methanol followed by 2 mL of water. The cartridges were washed with 2 mL of 0.1 M hydrochloric acid, then subsequently with 2 mL of methanol, 2 mL of water and, finally, with a mixture of methanol–conc. ammonium hydroxide–water 30:10:60 to eliminate acidic, neutral and some basic components of the urine. Elution was carried out with a mixture of methanol–conc. ammonium hydroxide–water 60:10:30. Eluates were evaporated to dryness under the stream of nitrogen at 70 ◦ C. The residues were re-dissolved in 100 ␮L of 50% aqueous methanol followed by 1.9 mL of 1% sodium hydrogen carbonate, pH of which was adjusted with 1 M aqueous sodium hydroxide to 10.5. The reconstituted samples were applied onto OasisTM HLB 60 mg SPE columns (reversed-phase sorbent containing both hydrophobic and hydrophilic groups, Waters), which were preconditioned with 2 mL of methanol followed by 2 mL of 1% sodium hydrogen carbonate solution (pH 10.5). The sample vials were rinsed twice with 1 mL of 1% sodium hydrogen carbonate solution and the washes were also applied onto the SPE columns, which were then washed with 2 mL of 35% methanol to eliminate interfering components. Adenine adducts were eluted with 1.5 mL of 80% methanol solution during 5 min, the eluates were evaporated to dryness under the stream of nitrogen at 70 ◦ C and the residues were re-dissolved in 60 ␮L of 30% aqueous methanol. After mixing shortly the solutions were transferred into 200-␮L glass vial inserts and analysed by HPLC/MS. Fig. 1. Hydrolytical cleavage of N3 adenine adducts from the DNA.

for toxicological studies and potentially also for biological monitoring of human exposure to some environmental mutagens and carcinogens (Shuker et al., 1984; Shuker and Bartsch, 1994; Prevost and Shuker, 1996; Casale et al., 2001; Yen et al., 1998; Bhattacharya et al., 2003). In our previous in vivo studies on mice exposed to styrene by inhalation we reported on the excretion of N7 guanine and N3 adenine adducts. They were formed by an attack of the nucleophilic nitrogen either at ␣- or ␤-carbon of the oxirane ring in SO molecule. Hence, four urinary adducts were identified, namely, 7-(2-hydroxy-1-phenylethyl)guanine (N7␣G), 7-(2-hydroxy-2-phenylethyl)guanine (N7␤G), 3-(2-hydroxy-1phenylethyl)adenine (N3␣A) and 3-(2-hydroxy-2-phenylethyl) adenine (N3␤A) (Vodicka et al., 2006; Mikeˇs et al., 2009). In this study we wished to reveal whether these urinary adducts are formed also in occupationally exposed humans at exposure levels close to the permissible exposure limit (PEL) value and what is their potential as biomarkers of effective dose.

2.4. LC-ESI–MSMS analysis The LC/MS system consisted of a Micromass triple quadrupole Quattro Premier XE (Waters Micromass, USA) interfaced with Agilent 1200 Series binary gradient pump (Agilent, USA) and CTC Analytics Autosampler (Switzerland). A silica based column HypersilGOLDTM 3.0 mm × 50 mm, 3 ␮m (Thermo Scientific, USA) was used for analyses. The mobile phase A was 10 mM ammonium acetate (pH 6.7) and the mobile phase B was methanol. The flow rate was 0.34 mL/min. For separation of adenine adducts the initial concentration, 25% of methanol, was kept constant for the first 0.33 min then changed linearly to 35% in 6 min and subsequently to 95% in another 1 min. After 2 min of isocratic elution with 95% methanol the initial mobile phase composition was re-adjusted. Total analysis time was 11.3 min. The analytes were detected by multi reaction monitoring (MRM) after electrospray ionization (ESI). The transitions in positive mode were m/z 256 → 136, which corresponds to the loss of PhCH2 CH2 O from (M+H)+ ions as the main fragmentation reaction. Continuous flow supply of the analytes solutions was performed to tune and establish MS/MS operating conditions. Capillary voltage was set to 0.19 kV, the source temperature to 110 ◦ C and the temperature of desolvation gas to 400 ◦ C. Desolvation gas flow was set to 1000 L/h. Collisions of the parent ions were performed using argon as a collision gas at a flow rate of 0.3 mL/min. Due to different operational optima for the ␣ and ␤ isomers two channels were used with different settings for each isomer. Thus, the collision energy was set to 15 and 19 eV and the cone voltage to 19 and 29 V for N3␣A and N3␤A, respectively. The linearity was verified by construction of a calibration curve in the concentration range from 0.5 to 5 pg/mL. The calibration samples were prepared from 1800 ␮L of blank urine spiked with 18 ␮L of appropriate standard solution. The calibration samples were taken through the same procedure as unknowns.

2. Materials and methods

3. Results and discussion 2.1. Human exposure Urine samples from 61 hand-lamination workers, 58 men and three women, of four reinforced plastics plants, which were occupationally exposed to styrene at various concentrations up to 286 mg/m3 , were collected at the end of the work shift. Additionally, ten of these lamination workers supplied also their urine samples collected next morning after the shift. All samples were analysed for mandelic acid as an indicator of styrene exposure. Of them, 28 end-of-shift samples with mandelic acid levels ranging from 68 to 1145 mg/g creatinine and all the 10 next-morning samples were selected for analysis of N3␣A and N3␤A. In addition, 22 samples of unexposed non-smoking volunteers, 17 men and five women, were used as controls. All samples were frozen immediately after collection and kept at −18 ◦ C until ˇ analysed. Mandelic acid and creatinine were determined by HPLC (Sperlingová et al., 2004; Schneiderka et al., 1993). Urine samples were provided to the investigators as anonymised specimens collected within a regular hygienic surveillance in the workplaces.

3.1. Method development A critical prerequisite for the urinary adduct determination is a sensitive and specific analytical method. In our previous study on styrene derived N7 guanine and N3 adenine adducts in mice urine we used an LC-ESI–MSMS method employing the main transition in the corresponding collisionally activated spectra. Sample pre-concentration was performed off-line using solid phase extraction (SPE). The limit of quantification for N7␣G and N7␤G was 30 and 20 pg/mL, respectively, whereas that for both N3␣A and N3␤A was ca.5 pg/mL, i.e., almost one order of magnitude lower (Mikeˇs et al., 2009). This difference was mainly due to a bet-

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ter retention characteristics of the adenine adducts so that these adducts could be pre-concentrated more efficiently than their guanine analogues. More favourable properties of the adenine adducts were due to several factors. Mainly, alkyladenines are more basic than alkyl guanines, e.g., pKa values for deprotonation of adenosine and guanosine are 12.4 and 10.0, respectively (Furumoto et al., 2001). Therefore, alkyladenines where retained on reversedphase columns even when washing them with alkaline solution (pH 10.5) for elution of acidic matrix components whereas alkylguanines were not retained under these conditions. Similarly, the pKa values of conjugated acids of adenosine and guanosine are 3.11 and 1.90, respectively (Chatterjee et al., 2006). So, adenine adducts may be retained on strong cation exchange columns at pH around 2 whereas guanine derivatives are not ionized efficiently at these pH values and cannot be therefore retained. Further, greater basicity of the adenines makes them more easily ionizable in positive ESI increasing their MS response 2–3 times as compared to alkylguanines. Finally, since MH+ ions of both N3␣A and N3␤A undergo partial spontaneous fragmentation in the ion source, exceedingly low setting of the capillary voltage at 0.19 kV could be used. At this voltage most matrix components were not ionized so that selectivity of the ionization was enhanced significantly. When double stranded DNA was reacted in vitro with SO at physiological pH, N7 guanine adducts accounted for nearly 90% of all alkylation products while, under the same conditions, N3 substituted adenines accounted for only 4–9% of the total alkylation (Koskinen et al., 2000, 2001b, Vodicka et al., 2002). However, in the urine of mice exposed to styrene, the ratio of N7 guanine and N3 adenine adducts was quite different, with N3␣A + N3␤A amounting to more than half of their guanine analogues (Mikeˇs et al., 2009). Hence, from the analytical point of view, urinary N3 adenine adducts rather than N7 substituted guanines seem to be promising as prospective biomarkers of the DNA damage caused by occupational exposure to styrene. Therefore, further attention was focused on the development of an improved analytical procedure for N3␣A and N3␤A. This was achieved by employing two pre-concentration steps, namely, SPE on a strong cation exchange column followed by SPE on a reversed-phase column that resulted in decreasing the limit of quantification to as low as 1 pg/mL for both N3␣A and N3␤A. Significantly variable matrix effects were observed among different urine samples spiked with the adenine adduct standards. To minimise these matrix effects, standard addition method was applied. 3.2. Expected level of the adenine adducts in styrene-exposed humans Presuming the same conversion of inhaled styrene to the urinary adenine adducts in mice (ConvAdA = 0.8 × 10−5 %) and humans, the expected amount of these adducts excreted in urine within 24 h (mAdA ) can be calculated as mAdA = ConvAdA × Dinh ×

M

AdA



MSTY

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where MAdA is the molecular mass of adenine adduct isomers N3␣A and N3␤A, MSTY is the molecular mass of styrene, Dinh is the dose of styrene absorbed by inhalation. An adult man exposed to styrene at 100 mg/m3 for 8 h (1.0 PEL) with a lung ventilation of 20 L/min and lung retention of styrene 55% (i.e., with total styrene uptake of 528 mg) should excrete approximately 104 ng of the adenine adducts per day. Value of the lung ventilation was estimated taking into account character of the hand-lamination work, which requires light to moderate physical activity (Derelanko, 2000). According to the experiment on mice no accumulation of adducts was considered because of their rapid release from the DNA (Koskinen et al., 2000; Mikeˇs et al., 2009). Assuming further an average diuresis of 2 L urine per day the expected concentration of N3␣A + N3␤A in the exposed human should be approximately 52 pg/mL. In this study we analysed 28 human urines (from 27 men and one women) with mandelic acid concentration ranging from 68 to 1145 mg/g creatinine corresponding to 8 h styrene exposure levels ranging from 17 to 286 mg/m3 . These values were calculated from the equation ˇ derived from literature data (Greim and Lehnert, 1998; Cern y´ et al., 1990; Guillemin and Berode, 1988):





mandelic acid mg/g creatinine at the end of shift



≈ 4 × styrene mg/m3



Samples were divided into two groups: (i) high exposure group with mandelic acid concentrations above 400 mg/g creatinine corresponding to styrene exposure exceeding the PEL of 100 mg/m3 , and (ii) low exposure group with mandelic acid concentrations below 400 mg/g creatinine. All members of the high exposure group were men. Average mandelic acid concentrations were 711 ± 238 mg/g creatinine and 232 ± 97 mg/g creatinine, corresponding to inhaled styrene concentrations of 178 and 58 mg/m3 for the high and low exposure group, respectively. At these exposure levels, expected urinary concentrations of N3␣A + N3␤A should amount to 92 pg/mL and 30 pg/mL, respectively. Both of these values are well above the limit of quantification of the analytical method described. 4. N3 Adenine adducts in human urine Sixty-one end-of-shift urine samples from hand-lamination workers were collected. In addition, next-morning samples were obtained from ten of these workers. All samples were analysed for mandelic acid as a major metabolite and a biomarker of exposure to styrene. Twenty eight end-of-shift samples with the highest levels of mandelic acid (68–1145 mg/g creatinine) and all ten nextmorning samples were then chosen for subsequent analysis of the adenine adducts by LC-ESI–MSMS. Samples were further divided into high and low exposure group with MA concentrations at the end-of-shift above or below 400 mg/g creatinine, respectively. Peaks co-eluting with authentic N3␣A and/or N3␤A were detected

Table 1 Urinary DNA adducts N3␣A and N3␤A (mean ± S.D.) for two exposure groups selected according to mandelic acid content as a biomarker of styrene exposure and the control group. Urine samples

n

Mandelic acid (mg/g creatinine)

Styrenea (mg/m3 )

N3␣A (pg/mL)

End-of-shift high exposure End-of-shift low exposure Next-morning high exposure Next-morning low exposure Unexposed

9 19 6 4 22

711 ± 238 232 ± 97 259 ± 185c 28 ± 35d –

178 ± 60 58 ± 39 204 ± 50 42 ± 23 0

1.8 1.0 0.9 0.7 0.9

± ± ± ± ±

1.1 1.0 0.6 0.6 0.9

N3␤A (pg/mL)

N3(A + N3(A (pg/mL)

1.0 ± 0.6 0.8 ± 0.4 0.5b 0.5b 0.6 ± 0.2

2.8 1.8 1.4 1.2 1.5

± ± ± ± ±

1.6 1.3 0.6 0.6 1.3

Findings, which were below the limit of quantification, were set arbitrarily to 1/2 this limit, i.e., to 0.5 pg/mL for both N3␣A and N3␤A. a 8 h exposure value calculated from mandelic acid levels using the equation: styrene (mg/m3 ) ≈ 0.25 × mandelic acid at the end-of-shift (mg/g creatinine). b Arbitrary value for negative findings. c Mandelic acid in the end-of-shift urine of the same subjects: 816 ± 198 mg/g creatinine. d Mandelic acid in the end-of-shift urine of the same subjects: 168 ± 91 mg/g creatinine.

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in 7 out of 9 samples of the high exposure group and in 6 out of 19 samples of the low exposure group. In the set of six blank urine samples taken from unexposed laboratory and office workers, five were negative but one showed quantifiable concentrations of both N3␣A and N3␤A. Therefore, an additional set of 16 control samples was taken for analysis. Positive findings were observed again. Altogether, the peaks co-eluting with N3␣A and N3␤A were found in nine and three samples, respectively, out of 22 control urines. Average values of N3␣A and N3␤A as determined by LC-ESI–MSMS are shown in Table 1. Mass chromatograms of representative samples from exposed and unexposed individuals are shown in Fig. 1. Only findings in end-of-shift samples of the high exposure group were significantly higher than those of the unexposed group (P < 0.05) (Fig. 2). Although adenine adducts N3␣A and N3␤A are known to depurinate rapidly from double stranded DNA in vitro (Koskinen et

al., 2000) it could not be excluded that their excretion in exposed subjects might have escaped detection as it started only postexposure. Therefore, 10 urine samples were collected for the adduct analysis also next morning after the shift. Of those, N3␣A was detected in two subjects, both having high mandelic acid level in the end-of-shift urine, whereas no N3␤A was found in any of the subjects. This finding provided evidence that human excretion of the adenine adducts was not overlooked due to improper time of sampling but is genuinely very low. In samples with no adenine adducts found the actual value of each adduct should be between 0 and the limit of quantification. Therefore, we assumed that it was 1/2 of the quantification limit, i.e., ca. 1 pg/mL for the sum N3␣A and N3␤A. The presence of N3␣A and N3␤A in the urine of unexposed humans is actually very unlikely, therefore, the peaks should be interpreted as analytical interferences rather than true adenine adducts. Although LC-ESI–MSMS is generally considered to be an analytical method of high structure specificity, interferences may occur at low concentrations due to some weak transitions in the spectra of matrix components and/or styrene metabolites co-eluting with the analytes investigated. As these interferences were present only in some urine samples they were probably coming from the diet. Because of occasional presence of interfering peaks in blank urine, the positive findings in the exposed urine cannot be attributed solely to the adenine adducts. In fact, no significant correlation was found between concentration of mandelic acid and adenine adducts in our exposed urine samples casting some doubts upon their association with the exposure, especially in the low exposure group. However, at least in the high exposure group, a part of each peak area should have arisen from the true adenine adducts. Then, even if the obtained values are likely to overestimate the true concentrations of N3␣A and N3␤A, they still represent maximum concentrations of these analytes possibly present in the exposed samples. Therefore, it can be assumed that average excretion of N3␣A + N3␤A within the high exposure group at a mean styrene level of 178 mg/m3 (8 h, constant level) amounted to not more than 3 pg/mL (the upper confidence limit of 2.8 ± 0.2 pg/mL; mean ± S.E.M., 95%). Under these conditions the expected value as based on the experiments on mice in vivo is approximately 92 pg/mL, i.e., 31-times higher than observed. This difference can be attributed to less efficient metabolic activation to SO and/or more efficient detoxification of SO in humans as compared to mice (Mendrala et al., 1993). These results indicate that the overall DNA damage caused by styrene via its reactive metabolite, SO, as reflected by excretion of rapidly depurinating adenine adducts, should be much lower in humans than in mice at similar exposure levels. However, biological effects such as mutagenicity and carcinogenicity need not necessarily be related to the overall DNA damage but may be initiated by specific DNA adducts, which are not easily repaired by cellular repair mechanisms. Although the urinary adenine adducts N3␣A and N3␤A as well as any other nucleobase adducts may reflect the DNA damage caused by styrene in vivo they are not promising as biomarkers of exposure or effective dose due to their very low levels in human urine. Moreover, their determination in human urine at such low levels is severely limited by matrix effects even when highly efficient separation together with a structurally specific detection such as MRM is used. Rapid removal of N3 adenine adducts from DNA in vivo prevents any cumulation in the organism so that these adducts can reflect only a short-term exposure to styrene (Mikeˇs et al., 2009). 5. Conclusion

Fig. 2. Mass chromatograms of human urine. Urine samples were spiked with N3␣A and N3␤A, 2 pg/mL each. (A) Typical sample from the control group; (B) positive sample from the exposed group; (C) positive sample from the control group.

A sensitive LC-ESI–MSMS method was developed for the determination of two urinary adenine adducts, N3(A and N3(A,

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