Specific DNA adducts induced by some mono-substitued epoxides in vitro and in vivo

Chemico-Biological Interactions 129 (2000) 209 – 229

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Mini-Review

Specific DNA adducts induced by some mono-substitued epoxides in vitro and in vivo Mikko Koskinen *, Kamila Plna´ Department of Biosciences at No6um, Center for Nutrition and Toxicology, Karolinska Institute, S-141 57 Huddinge, Sweden Received 1 August 2000; accepted 6 September 2000

Abstract Alkyl epoxides are important intermediates in the chemical industry. They are also formed in vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in many toxicology assays which has been associated with their covalent binding to DNA. Here aspects of the formation and properties of DNA adducts, induced by some industrially important alkenes and mono-substituted epoxides are discussed. These include propylene oxide, epichlorohydrin, allyl glycidyl ether and the epoxy metabolites of styrene and butadiene. The major DNA adducts formed by epoxides are 7-substituted guanines, 1- and 3-substituted adenines and 3-substituted cytosines. In addition, styrene oxide and butadiene monoepoxide are able to modify exocyclic sites in the DNA bases, the sites being in the case of styrene oxide N2- and O6-positions of guanine, N6-adenine as well as N4-and O2-cytosine. In vivo the main adduct is the 7-substituted guanines. The 1-substituted adenines have also shown marked levels, and these adducts should also be targets in biomonitoring of human exposures. Due to its low mutagenicity, 7-substituted guanines are considered as a surrogate marker for other mutagenic lesions, e.g. those of 1-adenine or 3-uracil adducts. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Biomarkers; DNA adducts; Epoxides; Mutagenicity

Abbre6iations: AGE, allyl glycidyl ether; BD, 1,3-butadiene; BMO, butadiene monoepoxide; CBI, covalent binding index; CYP, cytochrome P450; DEB, diepoxybutane; EBD, epoxybutanediol; ECH, epichlorohydrin; EO, ethylene oxide; HP, 2-hydroxypropyl; PO, propylene oxide; SO, styrene 7,8-oxide. * Corresponding author. Tel.: +46-8-6089245; fax: + 46-8-6081501. E-mail address: [email protected] (M. Koskinen). 0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 0 ) 0 0 2 0 6 - 4

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1. Introduction Epoxides, or oxiranes, are oxygen containing heterocyclic compounds. Due to the large ring strain associated with the three-membered ring they are reactive molecules. Because of their reactivity they are important intermediates in chemical industry, especially in polymer production. Wide-ranging industrial applications of epoxides have resulted also in considerable human exposure. In addition to direct exposure, humans are exposed to epoxides via metabolism of different alkenes. Compounds such as ethylene, propylene, butadiene (BD), styrene, vinyl chloride and acrylamide are metabolised to 1,2-epoxides by cytochrome P450-dependent (CYP) monooxygenases. The human exposure is of concern because the epoxides are alkylating agents in vivo being able to react with different nucleophilic centers of cellular macromolecules including proteins and DNA. DNA adducts in turn have shown considerable association with carcinogenic processes in vivo [1,2]. The adduct formation of the simple epoxides have been reviewed previously [3– 5]. This minireview updates the previous ones by focusing to some of the more recent data on the range and properties of these DNA adducts in vitro and in vivo. The article covers some important mono-substituted epoxides to which humans are directly exposed or those that are formed by metabolism after exposure to corresponding alkenes (Table 1). Ethylene oxide (EO), a non-substituted epoxide, is included in some cases for comparison. All the epoxides, or the alkenes, are agents with considerable occupational exposures. Some of them like BD are also environmental pollutants to which humans are exposed via tobacco smoke or car exhaust. The most likely route of exposure to these agents is by inhalation, although the possibility of dermal and oral absorption should also be considered [6–11].

2. Metabolism The formation of epoxides from alkenes, including, e.g. ethylene and propylene, is mainly mediated by CYP-dependent monooxygenases. This occurs by incorporatTable 1 The epoxides included in this study, and the agents to which humans are exposed occupationally or in the environment, resulting in the epoxide exposure Epoxide

Substituent to the epoxy moiety

Exposure

Ethylene oxide (EO) Propylene oxide (PO) Butadiene monoepoxide (BMO) Epoxybutane diol (EBD) Diepoxybutane (DEB) Styrene 7,8-oxide (SO) Epichlorohydrin (ECH) Allyl glycidyl ether (AGE)

–H –CH3 –CHCH2 –CH(OH)–CH2(OH) –CH(O)CH2 –C6H5 –CH2Cl –CH2–O–CH2–CH2CH2

Ethene, EO Propene, PO Butadiene, BD BD BD Styrene ECH AGE

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ing an atom of molecular oxygen into the substrate [12]. Even though this process is the first step in transforming lipophilic chemicals to excretable form, certain chemicals are activated by this to their ultimate carcinogenic form. Styrene is metabolized to styrene oxide (SO), mainly by CYP2B6 followed by CYP1A2, CYP2E1 and CYP2C8 [13]. Some of the styrene metabolizing cytochromes have been found to be polymorphic [14]. BD, with two double-bonds in its structure, shows a rather complex metabolic pathway. It is mainly oxidized by CYP 2E1 and 2A6 primarily to 3,4-epoxy-1-butene enantiomers (butadiene monoepoxide, BMO) [15], which is subjected to further metabolism. It can be oxidized to diastereomeric diepoxybutane (DEB) or hydrolyzed to 3-butene-1,2-diol via epoxide hydrolase. 3,4-Epoxy-1,2-butanediol (EBD) may be formed by hydrolysis of DEB or by oxidation of 3-butene-1,2-diol [16–19]. 1,2-Epoxides are relatively long-lived in aqueous solution at neutral pH, the half-lives of hydrolysis at 37°C being of the order of 50–200 h [3,20]. The half-lives in vivo are some 100 times shorter, showing that the major detoxification pathways are enzymatic. Metabolism of epoxides generally leads to intermediates that are considerably less reactive than the parent compound. One detoxifying pathway of epoxides is the addition of water to form 1,2-diols, which are of low reactivity, the reaction being catalysed by epoxide hydrolase [21,22]. Several distinct forms of epoxide hydrolase have been identified, which differ in physical properties and substrate preferences. The extent to which epoxide hydrolase is involved in human metabolism of the studied epoxides is not clear, neither is the possible effect of the polymorphism of this gene. Still, this pathway was shown to be the major detoxification pathway of SO in humans [23,24]. Another possible pathway is inactivation by glutathione S-transferases, forming glutathione S-conjugates [25,26]. These conjugates are generally degraded in liver and kidney and excreted in urine as the corresponding mercapturic acids (N-acetylS-cysteine conjugates) [22]. The importance of this pathway in humans is indicated by the presence of S-hydroxyethylcysteine in the urine of EO exposed hospital workers [27]. Genetic polymorphism in several glutathione S-transferase genes has been found, which may cause differences between individuals in this metabolic pathway [28].

3. Reactions with nucleic acid constituents Mono-substituted epoxides have two electrophilic carbons, i.e. a- and b-carbons. The site of nucleophilic attack on alkyl epoxides under physiological conditions is primarily at the less substituted, sterically more accessible, b-carbon. The substituted a-carbon is expected to be less reactive, owing to steric hindrance and the electronic effects of the substituent. Epoxides with a vicinal aromatic (SO) or vinyl group (BMO) can, however, react through the both carbons because the substituent increase the positive charge at the a-carbon. Because a-carbon is asymmetric two diastereomeric products are therefore formed through nucleophilic attack on either carbon [3,5].

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The site of alkylation of the DNA constituents is mainly determined by the ionic character of the substrate [29]. Thus, alkyl epoxides that are not able to stabilise an ionic charge to any great extent, like aliphatic alkyl epoxides, react predominantly at ring nitrogen positions in DNA bases. Under physiological conditions the main alkylation sites are 7-guanine, 1- and 3-adenine, and 3-cytosine (Table 2). In contrast, SO and BMO modify also exocyclic groups [30–34]. The reaction mechanisms of nucleoside alkylation have been studied using optically active epoxides. In the case of ring-nitrogen substitution, the reaction through the b-carbon has been found to follow direct displacement by SN2 type of reaction mechanism [30,31,33,48]. In contrast, under neutral conditions the exocyclic sites open the epoxide in SO only at the a-carbon, resulting in both inverted and retained stereochemistry, indicating prominent SN1 type of nucleophilic attack [30,31,33]. It has been concluded that the exocyclic amino groups involve substrate ionisation that decreases in the order guanine \adenine\ cytosine, correlating inversely with the pKa values of those nucleic acid bases [33]. Mainly mono-substituted products are obtained by the treatment of nucleic acid constituents with epoxides. Bis-substituted deoxyguanosines have been detected by SO alkylation involving N2- and O6-positions or N2-and 1-positions [54]. Also bis-alkylation involving base and phosphate has been detected [55]. Phosphate alkylation in nucleotides has been detected for AGE, PO and SO [38,41,55]. When a dinucleotide dGpdT was treated with SO no phosphotriesters were detected in the intervening phosphate suggesting that they are not expected to be formed in DNA [55]. Epichlorohydrin (ECH) differs from the other epoxides under study by being a bi-functional alkylating agent [39,40]. It is susceptible to a second SN2 displacement reaction via the loss of chlorine. For adenine alkylation, it is proposed that the epoxide first undergoes ring opening by the 1-position, since it is the most nucleophilic nitrogen. Cyclisation and loss of HCl, via the attack of N6 on the carbon carrying the chlorine results in formation of the 1,N6-2-hydroxypropanoadenine adduct [39]. A similar product has been found after reaction of deoxyadenosine with cyanoethylene oxide, another bi-functional agent [56]. For the adduct studies, properly characterised standard compounds are needed. To prepare different standards in sufficient amounts, some adjustments of the reaction conditions are needed. The reaction under neutral pH usually leads mainly to 7-guanine substitution through the b-carbon in relatively high yields [5]. Reaction at glacial acetic acid shows also preferential 7-alkylation, however, in the case of SO favouring reaction at a-carbon [30]. When the reaction is performed under alkaline conditions, i.e. above the pKa-value of the 1-position of guanine, N2- and 1-alkylated products are obtained in higher yields [55,57]. The O6-alkylated standards are best obtained by reaction of 2-amino-6-chloropurine with the proper sodium alkoxide [5]. The optimisation of reaction conditions for preparation of the standards of the other nucleobases than guanine is rather little examined with the currently studied epoxides. In the case of adenosine, glacial acetic acid has shown to favour the ring-nitrogen alkylation through the a-carbon of SO, similarly as for 7-guanosine alkylation [31]. The 3-alkylated thymidine monophosphates can be

– – – – a a – –

ba b b b a,b a,b b b

b – – – a,b – – –

O6 b b bd b a,b a,b b b

N1

Adenine

bb bb bd bb a,bb a,bb bb bb

N6 b b – b a,b a,b b b

N3 b b b b a,b a,b – –

N3

Cytosine

– – – – a,b b – –

O2 – – – – a – – –

N4

b b b – a,b a,b – –

N3

Thymine

bc bc bc bc ac,bc ac,bc – –

N3

Uracil

[35,36] [37,38] [39,40] [41] [30,31,33,34,42] [32,43–49] [50–52] [50–53]

References

b

a

b, reaction through b-carbon of the epoxide; a, reaction through the a-carbon. In the case of EO a- and b-carbons can not be distinguished. Secondary products formed from by the Dimroth rearrangement from the 1-alkylated adenine. c Secondary products formed by deamination from the 3-alkylated cytosines. d Data on 1- and N6-adenine of ECH represent formation of the 1,N6-2-hydroxypropano adduct.

EO PO ECH AGE SO BMO DEB EBD

N2

N7

Guanine

Table 2 Binding sites of the epoxides in nucleobases formed by the reaction of the epoxide and the nucleic acid constituent in vitro under physiological pH and temperature

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Table 3 In vitro stabilities (half-lives) of the different adducts in double-stranded DNA, at neutral pH and 37°C

EO PO ECH AGE SO (a/b) BMO BDE EBD

7-Guanine (h)

3-Adenine (h)

1- to N6-adenine rearrangement (h)

3-Cytosine deamination (h)

References

75 120 72 38 51/51 48 30 31

10–15 – – 20 10/21 – – –

– 221 – 150 – – – –

– 53 – 48 – – – –

[36] [38] [64] [40,41] [65] [48] [52] [52]

obtained in high yields at alkaline conditions, as well as, 3-alkylated uridine monophosphates [34,46,55].

4. Reactions with DNA in vitro In DNA, EO and the mono-substituted alkyl epoxides all modify preferentially the 7-position of guanine mainly due to the high nucleophilicity and sterical availability of the position [3,5]. Other primary adducts of alkyl-epoxides are those at 1- and 3-position of adenine and 3-position of cytosine [35,38,41,49,51,58]. In the case of SO, the spectrum is wider due to the reactive a-carbon, including also N2and O6-positions in guanine, N6-adenine and N4-cytosine [34,59–61].

4.1. Stabilities of the adducts Ring nitrogen-substituted products are all unstable in some fashion; thus, their possible biological effects might be influenced by chemical transformations secondary to the initial DNA alkylation. The secondary transformations are generally qualitatively similar in nucleosides and nucleotides as in DNA, even though the rates may differ considerably. Examples of such instabilities are the facile depurination of 7-alkyl-deoxyguanosine and 3-alkyl-deoxyadenosine, and the imidazole ring opening of 7-alkyl-deoxyguanosines [62]. These reactions are acid- and alkalicatalysed, respectively, but occur even at physiological conditions. The depurination is due to the quaternary nitrogen sites, which lead to cleavage of the glycosyl bond, to regain the aromaticity. The 3-alkylated adenine adducts are considerably more labile than those of the 7-guanines (Table 3). Since both of these secondary lesions can persist in DNA for extended periods, they may contribute to induction of biological consequences. On the other hand, the lability of the 7-guanine and 3-adenine adducts can also be useful in applying them as noninvansive biomarkers of exposure by monitoring their amounts in urine [63].

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Other secondary lesions are products at the 3-position of uracil, which have been shown to originate from deamination of the corresponding 3-cytosine adduct [34,38,41,58,66]. Furthermore, alkylations of N6-adenine have been shown to originate from a Dimroth rearrangement of 1-substituted adenine, facilitated by the adjacent hydroxyl group [37]. Direct alkylation at N6-adenine occurs primarily after substitution at the a-carbon of epoxides containing electron-withdrawing substituents, such as SO and BMO [47,61,66]. In addition to the Dimroth rearrangement, 1-alkyl-adenines, especially in the case of SO-adducts, can undergo deamination to the corresponding hypoxanthine adducts [61].

4.2. Relati6e amounts of the adducts The adducts are generally formed in a time- and concentration-dependent manner. However, because of the differing stabilities of the adducts, the relative proportions of the adducts at different sites may depend on the length of exposure. 7-Guanine and 3-adenine adducts reach an apparent steady-state level while the level of the chemically more stable adducts continue to increase. Table 4 shows the amounts of other adducts relative to that of the 7-position of guanine. In doublestranded DNA, 75 – 90% of total alkylation takes place at the 7-position of guanine, and 4 – 8% at the 3-adenine. Thus, a vast majority of the alkylation occupies labile sites that lead to apurinic sites. The 1-position of adenine and 3-cytosine are highly nucleophilic sites that do not react extensively in DNA because of steric hindrance. The sum of 1- and N6-substituted adenines have been found in the range 4–14% for aliphatic alkyl-epoxides relative to the 7-alkylation. The sum of 3-substituted cytosines/uracils has been found in the range 2 – 6% of 7-substituted guanine (Table 4). The yields of 1-AGE-adenine and 3-AGE-cytosine (including 3-AGE-uracil) in single-stranded DNA were 19 and 6 times higher, respectively, as compared to double-stranded DNA [38]. This difference is expected since the 1-position of adenine and the 3-position of cytosine are involved in the Watson–Crick base pairing. The corresponding difference for the BMO adducts between double- and single-stranded DNA was reported to be in order of 10–20 times [58]. In the case of 1-adenine alkylation by SO, also the deaminated products are found to constitute a considerable proportion of the total alkylation [61]. The distribution of the adduct originating form those of 1-adenines has been found to differ in nucleosides, single-stranded DNA and double-stranded DNA in favour of formation of b1-SO-deoxyinosine with increased structural complexity [61]. This indicates the importance of the deaminated fraction in in vivo. In the case of the SO- and BMO-alkylation different regioisomers are formed. In DNA, the ring-nitrogens open primarily the b-carbon of SO and BMO, being in accordance with the alkylation of free nucleic acid constituents. For the 7-alkylguanines the reaction through the b-carbon predominates only slightly [34,49,58,65]. However, in the case of the 3-alkyl-adenines, the a-isomer predominates, being in contrast to the alkylation of free adenine [49,65]. The reason for the reversed reactivity order for adenine within DNA was suggested to be related to

100 – 10 10 1 –

100 – 10 3.5 2 –

PO [38] 100 – 11 14 6 –

AGE [41] 100 – – 9c 4 –

ECH [40,64]

100 4.5 10 16d 5 2

SOb [67]

b

The values given are relative to the 7-substituted guanines. Different geometrical isomers are not separated in the case of SO and BMO. c Data on 1- and N6-adenine of ECH represent formation of the 1,N6-2-hydroxypropano adduct. d Includes the deaminated 1-adenine adduct. e Only 1-adenine [53]. f Only N6-adenine [59].

a

7-Guanine N2-Guanine 3-Adenine 1-/N6-adenine 3-Cytosine/uracil N4-Cytosine

EO [35]

100 – 15 25e/0.5f 1.5 –

BMOb [53,58,68]

Table 4 Relative amounts of different adducts formed by the reaction of the epoxide and double-stranded DNA in vitro, at neutral pH and 37°Ca

100 – 11 – – –

EBD [68]

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steric constraints in the double-stranded DNA [49]. Moreover, the 3-alkylation of adenine in DNA is in contrast to the corresponding reaction of free nucleosides in which no such products are observed. In DNA, the 3-position of adenine is not involved in hydrogen bonding and is sterically exposed in the minor groove of a-helical DNA thus being accessible for alkylation [58]. In contrast, in free nucleosides the site may be blocked by intra- or intermolecular hydrogen-bonding [69]. Rate constants for reaction with DNA can be determined in in vitro experiments to compare the reactivity of different epoxides [70,71]. In Table 5 are given the second-order rate constants for the reaction of some epoxides with 7-position of guanine in DNA in vitro. The rate constant for the reaction of AGE with 7-guanine in DNA [41] is about twelve and three times lower than those for EO and PO, respectively. Thus, the rate of reaction seems to decrease with increasing length of the substituent on the epoxide, as it has been previously observed for other related epoxides [72,73]. The reactivity of ECH towards DNA is about twice that of PO. This is in accordance with the relative reactivity of the two compounds towards model nucleophilic compounds such as water and ammonia [3]. Based on its relative stability in DNA and high concentration relative to the other adducts formed, 7-substituted guanine would be a suitable marker of epoxide exposure. Although 1-substituted adenine is present at lower levels than 7-substituted guanine, it is considerably more stable and this adduct could therefore be a suitable alternative, particularly if the rearrangement product, N6-substituted adenine, could be analysed simultaneously. The stable (and biologically probably more important) 3-uracil adduct is yet another alternative biomarker, although its levels are expected to be considerably lower. Products with thymidine are expected to be present at very low levels [5,46].

Table 5 Chemical reactivity of epoxides in vitro for the 7-position of guanine in DNA and corresponding CBI for rat or mouse liver DNA after a single i.p. dose

EO PO AGE ECH Styrene a

kDNA (7-guanine)×104a (l/g DNA per h)

CBI (nmol 7-guanine/mol dNp per mmol/kg body weight)

Reference

0.96 0.25 0.08 0.6 0.18c

11 0.6 0.04b 0.2 0.2b

[36,74] [75] [76] [64,77] [78]

The rate constants were calculated as in [70]. Calculated for mouse liver DNA. c The rate constant of SO, M. Koskinen, unpublished. b

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5. Specific DNA adducts in vivo DNA adduct formation of chemical carcinogens or their reactive metabolites is considered to constitute an initial step of carcinogenesis [1]. The adduct level represents an integration of exposure, absorption, distribution, metabolism and reactivity of carcinogen; chemical stability of adduct; the action of repair processes; and cell turnover. As long as the passive uptake, transport mechanisms or enzymatic processes are not saturated, inhibited or induced a linear dose-response relationship for adduct formation is expected after in vivo exposures. Consequently, saturation or induction of different processes involved in formation and repair of DNA adducts are likely to have important implications for mutagenesis and carcinogenesis, particularly when exposing experimental animals to high doses and using data from such studies in cancer risk assessment [79,80]. In addition, deviation from linearity could take place if the exposure is causing toxicity or proliferation to the studied cells. The net result of various toxicokinetic parameters has been termed biologically effective dose (molecular dose) — that is, the interaction of the carcinogen with critical cellular targets [81]. Recent studies in animals [82] and humans [83] suggest that levels of DNA adducts in tissues are indeed reflective of carcinogen doses.

5.1. Adducts in experimental animals 5.1.1. 7 -Substituted guanines The formation and stabilities of the alkyl epoxide-induced DNA adducts have been studied in rodents in order to understand the role of specific adducts in in vivo models. Following single and repeated exposures of rats and mice to EO demonstrated similar accumulations of 7-(2-hydroxyethyl)-guanines in both target and various non-target tissues, except in testis where lower levels were observed [84]. This is obviously due to the rapid and even distribution of EO to all tissues except testis, and the ability of EO to act as a direct alkylating agent [84]. 7-(2-Hydroxyethyl)-guanines were disappeared slowly from mouse kidney and rat brain and lung, being consistent with the loss of the adduct by chemical depurination. However, in the other organs studied more rapid removal was observed indicating active repair [84]. After exposure to PO, another directly alkylating agent, PO-substituted 7-guanines were found in considerably higher levels in target tissue (nose) when compared to non-target tissues including lungs, lymphocytes, spleen, liver and testis [85]. By exposure of 500 ppm of PO for 20 days the alkylation at the respiratory nasal epithelium was up to 98 PO-adducts per 106 nucleotides. The rate of elimination of the 7-substituted guanine was about the same in all examined tissues, corresponding closely to the spontaneous rate of depurination of this adduct, thus excluding the active repair of the 7-PO-guanine adducts [85]. The higher adduct level in the respiratory tissues is therefore due to a higher dose of PO deposited in this organ. For epoxides that are formed by the CYP-systems from alkenes the adduct formation is somewhat more complex event. It is expected that adduct formation

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would be higher in the organs where the metabolic activation is taking place. Indeed, in mice liver where the CYP activity is highest, slightly higher 7-BMO-guanine levels were detected after BD inhalation, as compared to lung and kidney [86]. But the difference was only slight, suggesting that the DNA-reactive metabolites circulate in the blood and reach steady-state concentrations over time [86]. By the exposure of rats and mice to BD, the two isomers of the 7-BMO-guanines are formed to similar extent [68]. However, differences have been observed in the levels of different enantiomers which may be related either to enantioselective repair or formation of the reactive metabolites [87]. The 7-trihydroxybutyl-guanine adducts formed from EBD (or DEB) constitute the highest proportion of the BD derived adducts in vivo [52,86]. The different enantiomers of trihydroxybutyl-adducts varied in the manner that adducts derived from RR and SS enantiomers of EBD was twice the level from RS and SR enantiomers [88]. Contrasting results have been reported, suggesting the main adduct being the RS isomer of EBD, which was explained by the authors to be due to the differences in the production rates of RRor RS-BDE isomers among different mouse strains [52]. As in the case of PO, the persistence of 7-trihydroxybutyl-guanine adducts was similar to the half-life of spontaneous in vitro depurination [88]. Slight species differences were observed between mice and rats, the persistence of being somewhat greater in mice. This suggests more active repair of these adducts in rats [88]. After single i.p. injection of styrene, Pauwels et al. [78] determined the 7-guanine adducts of SO in various mice tissues. For doses up to 4.35 mmol/kg b.w., adduct levels up to 6.3 of 7-guanine adducts per 107 nucleotides were detected, with a clear dose-response relationship. The adducts were most abundant in the lungs, ca. 30% more than in liver and spleen. The higher adduct level in lungs could be a result of high CYP-activity converting styrene to SO and for lack of epoxide hydrolase activity in that organ. The comparison of the adduct levels induced by different epoxides is difficult mainly due to the large variation in the protocols used in animal studies. The covalent binding indices (CBI), adduct level per unit exposure dose, has been used to compare chemical carcinogens with respect to their capacity to alkylate macromolecules in vivo. The CBI of different epoxides at the 7-position of guanine in liver DNA of rodents exposed to single intra peritoneal dose (assuming linear dose – response relationship for adducts formation) is shown in Table 5. Differences in adduct levels formed by the studied epoxides in vivo could be attributed, to a certain extent, to differences in rates of alkylation at the nucleophilic sites in DNA. The difference between EO and PO is higher than what can be explained solely by different reactivity of these two compounds toward 7-guanine. A higher rate of detoxification of PO in rat liver is a likely contributing factor. The observed difference in levels of PO versus EO adducts lasted even after repeated exposures by inhalation (20 days), at conditions when the steady-state level was most likely achieved. Under such circumstances, the concentration of the 7-guanine adduct in lung and spleen from 500 ppm PO [85] was about 2 times lower than the concentration of the 7-guanine adduct found in lung and spleen of rats exposed to 100 ppm EO [89] for the same period of time, i.e. EO being 10 times more efficient than PO.

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Comparison of styrene to the epoxides in Table 5 is hampered by the need for metabolic activation to SO. SO and PO have similar reaction rates in vitro but PO gives higher CBI value, which is probably related either to the slow formation of SO or faster detoxification of SO compared to PO. Also, in spite of higher chemical reactivity, the capacity of ECH to alkylate rat liver DNA [64] (Table 5) is lower than that of PO, indicating a faster detoxification of ECH compared to PO. The difference between ECH and PO in alkylating capacity in vivo was confirmed in studies of haemoglobin adducts in rats [77]. Among studied compounds, AGE has the lowest ability to alkylate 7-guanine in DNA in vivo [76], as indicated by its lowest in vitro reactivity towards this position in DNA [41]. In another study, binding of glycidyl ethers to haemoglobin of mice was studied [20]. The haemoglobin binding indices, 1.1–1.2 pmol/g globin for AGE and butyl glycidyl ether and 1.3 pmol/g globin for phenyl glycidyl ether and cresyl glycidyl ether per kg body weight, were similar to that of PO (1.4 pmol/g globin) and 5–6-fold lower than that of EO (7 pmol/g).

5.1.2. Substituted adenines and cytosines Because of the instability of the 7-substituted guanines, they are reflective of a rather recent exposure. In the case of a chronic exposure, chemically more stable 1or N6-substituted adenines and 3-substituted cytosines or uracils should be the target, since they should be accumulating in the absence of active repair. More low-dose long-term animal studies would be needed to better characterise the accumulation of these adducts and thus to predict the adduct relevant for the human studies. Recently, 1-alkylated adenines have been detected by the 32P-postlabelling assay in rodents after conversion to the N6-alkylated adenines by the Dimroth rearrangement [38,53,90]. In rats exposed to 500 ppm of PO by inhalation during 20 days, 1-hydroxypropyl (HP)-adenine was detected in nasal epithelium, lung and lymphocytes [38]. N6-HP-adenine, on the other hand, was found only in the tissues of the nasal cavities. The highest level of 1-HP-adenine (2.0 adducts per 106 nucleotides; i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium, which also represents the major target for tumour induction in the rat following inhalation of PO. The levels of this adduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium, corresponding with the formed adduct levels of 7-HP-guanine in the same tissues [85]. In rats sacrificed 3 days after cessation of exposure, no significant decrease of 1-HP-adenine was observed, indicating a very slow repair of this adduct [38]. Also, the formation of the N6-adenine [87] and 1-adenine [53] adducts of BMO and the 1-trihydroxybutyl-adenine adducts [90] in rats exposed to BD by inhalation have been identified. Similarly to 7-substituted-guanines and haemoglobin adducts the 1-trihydroxybutyl-adenine is formed to higher levels than the corresponding BMO adducts [53,68,91]. 1-BMO-adenine adduct level was ca. 40% of the 7-BMOguanine level being somewhat more than the BMO-proportion in the in vitro treated DNA [53]. N6-BMO-guanine was ca. 0.5% of 7-substituted guanines [87]

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corresponding to proportions in in vitro BMO-treated DNA. The amount of N6-BMO-adenine was the same immediately after the exposure and 3 weeks later indicating chemical stability and lack of efficient DNA repair machinery for the N6-adduct [87]. Recently, the 1-SO-adenine adduct has also been detected in mice lungs after inhalation of styrene [67]. The b1-SO-adenine adducts levels were ca. 3% of the level of the b7-SO-guanines, being slighly lower than the proportion in in vitro SOtreated DNA. Also the O6-SO-guanines have been identified by the 32P-postlabelling methods after i.p. injection of styrene [78] or by inhalation [92]. 3-HP-uracil have been detected in the respiratory nasal epithelia of PO-exposed rats with concentration of 0.02 adducts per 106 nucleotides (0.02% of 7-HP-guanine), suggesting repair of the cytosine and/or uracil adducts. Further support for this hypothesis was obtained by incubation of PO-treated DNA with a protein extract from mammalian cells. In this experiment 3-HP-cytosine, but not 3-HPuracil or 1- and N6-HP-adenine was repaired [38]. Furthermore, experiments with a bacterial uracil glycosylase indicated that this enzyme was not involved confirming the presumption that 3-alkyl-cytosines are not expected to be repaired by this enzyme [38].

5.2. Human adducts The adduct studies in humans concerning the epoxides under study are still quite few, but, especially due to the recent advances in the 32P-postlabelling assay, the data are amounting. Particularly, DNA-adduct data relative to polymorphisms of xenobiotics metabolising enzymes can be expected in the near future, revealing the individual susceptibility to the DNA adduct formation after the exposure. In populations exposed to relatively high concentrations of styrene significant increases of levels of DNA adducts, that were assigned as O6-substituted guanines [93– 95] and N2-substituted guanines [96], have been reported. In lymphocytes of laminators the mean O6-guanine adduct level was 5.4/108 normal nucleotides as compared to 1.0/108 in controls [94]. However, the identity of these adducts have later raised some doubts [92]. The mean adduct level reported by Horvath et al. [96] for N2-guanine adduct was 16/108 with a linear relationship between dose of styrene exposure and the adduct level. More recently, by using 32P-postlabelling/HPLC assay Zhao et al. [97] reported the 1-trihydroxybutyl-adenine adducts in lymphocytes of humans occupationally exposed to BD. The mean adduct level in the exposed workers was 4.5 adducts/109 normal nucleotides as compared to 0.8/109 in controls. Even though being the main adduct in vitro and in animal studies, the 7-guanine adducts of the epoxides have been difficult to detect in humans. 7-(3-Chloro-2-hydroxypropyl)-guanine adducts after exposure to ECH has been reported, the adduct levels ranging from 1 to 7 adducts/109 nucleotides [64]. The levels of 7-(3-chloro-2hydroxypropyl)-guanine detected were 1–2 orders of magnitude lower than the 7-methyl- and 7-(2-hydroxyethyl)-guanine reported in white blood cells of both non-smokers and smokers [98,99].

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6. Mutagenic aspects of specific adducts Several studies have indicated that only certain DNA alkylation products contribute to the mutagenic or carcinogenic activity of alkylating agents. For instance, it has been found that O6-alkyl-guanines are quantitatively associated with carcinogenesis or mutagenesis [100,101], whereas low molecular weight adducts at 7-position of guanine are generally not promutagenic [102]. Secondarily formed apurinic sites and ring-opened products on the other hand are potentially mutagenic lesions [103]. Most epoxide-alkylation in DNA has been shown to take place at 7-guanine and 3-adenine, leading to potentially mutagenic apurinic sites. The 7-guanine and 3-adenine adducts are expected to result in GC“ TA and AT “ TA transversions, respectively, since DNA polymerase preferentially adds an adenine opposite to an apurinic site [103]. Such mutations have indeed been found in SO-treated hypoxanthine-guanine phosphoribosyl transferase (hprt) mutant clones [104] and in POtreated Salmonella hisG46 and hisG428 [105]. AT “ TA transversions have also been identified at the hprt locus in mice splenic T cells exposed to BD, whereas exposure to BMO and DEB produced more GC “TA transversions [106]. It has been shown that in genomic DNA the steady state of apurinic/apyrimidic sites is 1 lesion per 105 nucleotides [107]. In humans, the adduct levels induced by the epoxides studied could be expected in level up to few adducts per 108 nucleotides and in experimental animals few adducts per 106 nucleotides. Therefore, the mutagenic role of the apurinic sites originating from the 7-guanine or 3-adenine adducts induced by these epoxides can be considered rather small, especially because the apurinic/apyrimidic sites are constantly being repaired. Even though formed to lower extent, substitution at a base-pairing sites of DNA can be expected to be more mutagenic as compared to the 3-adenine or 7-guanine adducts. The dominating type of SO-induced hprt-mutation was the AT “ GC transition [104] and short term animal studies on BD have shown the mutations at the AT base pairs to be the predominant ones [108–111]. These mutations are likely related to 1- or N6-alkylation of adenine residues. The AT“ GC transition was observed in a site-specific mutation study in which a SO adduct at N6-adenine was inserted in N-ras gene codon 61 [112]. However, the N6-adenine adduct showed a rather low miscoding potential [112], probably because the adduct has still the possibility for base-pairing with thymine residues. The same transition was also observed in a study by Carmical et al. [113] where RR enantiomer of BDE was inserted at the N6-position of adenine within the N-ras codon 61. Interestingly, the corresponding SS enantiomer yielded exclusively AT“ CG mutations [113]. It appears that the N6-adenine adducts are not responsible for the mutagenesis associated with the exposure BD or styrene metabolites. More likely mutagenic candidates are the 1-adenine adducts, or the corresponding deaminated 1-hypoxanthine adducts [61], since they occupy a central Watson–Crick base pairing site disrupting the normal hydrogen-bonding. In the case of the BD metabolites, DEB is 100-fold more mutagenic than BMO and is probably involved in the BD-induced carcinogenicity [114]. This might be

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related to the cross-linking ability of DEB [115,116], or to the high levels EBD adducts originating from DEB. N2-guanine adducts of BMO and BDE were found to be an order of magnitude more mutagenic than the N6-adenine adducts in recent a site-directed mutagenesis study [117]. The different relative mutagenic potency of various stereoisomers was evident, but no distinct mutational signature of the N2-guanine adducts were observed. Deamination of 3-cytosine adducts may also have a marked role in the mutagenesis induced by the epoxides studied. For propylene oxide it has been suggested that 3-HP-uracil is a mutagenic lesion [118] leading to GC“ AT and to minor extent GC “TA and GC “CG mutations [119]. Recently it was shown that using protein extracts from mammalian cells that enzymatic repair exists for removal of 3-HP-dCyd but not for the corresponding uracil adduct, and that the uracil glycosylase is not working on the adducts [38]. Furthermore, a mutagenicity study applying a site-specific incorporation of a 3-hydroxyethyluridine adduct in a 55-nucleotide template suggested that the adduct may be a critical mutagenic lesion induced by EO, leading to GC“ AT and GC“ TA mutations [120].

7. Conclusions 7-Guanine and 1-adenine adducts could be useful as biomarkers of exposure to the studied epoxides. The major advantage of 7-substituted guanines is their high concentration relative to the concentration of other adducts formed. But because of their lower mutagenicity they can be mainly used as a surrogate marker for other promutagenic adducts. Although the levels of 1-substituted adenines in DNA are lower than that of 7-substituted guanines, they represent a feasible alternative due to persistence in vivo (shown for propylene oxide) and the high specificity and sensitivity of 32P-postlabelling analysis. Further, the 1-adenine adducts are interesting since they appear to have an important role in mutagenicity of the epoxides. Another lesion by the epoxides that should be considered in the adduct studies in vivo is the 3-substituted uracil. This is mainly because of its persistence and obvious mutagenicity.

Acknowledgements Thanks are due to Professor Kari Hemminki for critical reading of the manuscript. The work was supported by the Swedish Council for Work Life Research and by the European Communities, QLK4-1999-01368.

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