DRUG METABOLISM REVIEWS, 31(1), 175–193 (1999)

THE MECHANISM OF BIOACTIVATIONOF N-NITROSODIETHANOLAMINE*

RICHARD N. LOEPPKYDepartment of ChemistryUniversity of MissouriColumbia, Missouri 65211

I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

II. IS NDELA ACTIVATED BY β-OXIDATION? . . . . . . . . . . . . . . . . . . . 177

III. REINVESTIGATION OF THE α-OXIDATION PATHWAY FORNDELA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

IV. OTHER BIOACTIVATION PATHWAYS . . . . . . . . . . . . . . . . . . . . . . . . 187

V. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

I. INTRODUCTION

N-Nitrosodiethanolamine (NDELA) (Structure 1), is one of the most wide-spread nitrosamines in the human environment because it forms from dietha-

* This paper was refereed by Sidney D. Nelson, Ph.D., School of Pharmacy,University of Washington, Seattle, WA 98195-7631.

175

Copyright 1999 by Marcel Dekker, Inc. www.dekker.com

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176 LOEPPKY

STRUCTURE 1

nolamine, triethanolamine, and various derivatives thereof which are used in agreat many formulations ranging from cosmetics and personal care items to met-alworking fluids [1–9]. Despite the fact that it has been demonstrated to be apotent carcinogen in several species of animals by means of dose-response studies[10–13], its mode of carcinogenic activation has remained relatively obscure.The purpose of this review is to summarize various hypotheses related to itsmode of bioactivation and how new data discriminate between them and pointthe direction of further investigation.

Most nitrosamines are believed to be activated by cytochrome P450 (CYP)-mediated α-hydroxylation as depicted in Scheme 1 [14,15]. α-Hydroxynitrosam-ines (1) have been synthesized and shown to decompose rapidly in aqueous solu-tion with evolution of N2, which is assumed to arise from a diazonium ion inter-mediate (2) [16,17]. α-Acetoxynitrosamines are often used as relatively stableprogenitors of α-hydroxynitrosamines and are helpful in testing hypotheses re-garding the nature of metabolites, as well as protein and nucleotide adducts ex-pected from metabolically generated diazonium ions [18–20]. Numerous studieshave revealed a similarity between in vivo nitrosamine-produced alkylation prod-ucts and those derived from α-acetoxynitrosamines in vitro and in vivo, therebystrengthening the view that the bioactivation of many nitrosamines involves α-hydroxylation [21,22].

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MECHANISM OF BIOACTIVATION OF NDELA 177

II. IS NDELA ACTIVATED BY b-OXIDATION?

Work on NDELA has presented several challenges to this metabolic format.NDELA is unusual for a nitrosamine, in that a high percentage of the dose isexcreted in the urine unchanged, even at low doses [23–25]. Compared to othernitrosamines, NDELA results in very low levels of 14C incorporation into DNA[23,26]. NDELA is not mutagenic in the standard microsome-mediated Amesmutagenicity assay [27,28]. Other studies have failed to observe products ex-pected of the metabolic α-hydroxylation of NDELA using either microsomalpreparations or primary rat hepatocytes [29]. The latter findings have been usedto explain the failure of NDELA to produce a mutagenic response in microsome-mediated assays.

The metabolic disposition of NDELA (Scheme 2) has been investigated byAiroldi and co-workers, who found a single urinary metabolite, N-nitroso-N-2-hydroxyethylgycine (NHEG) [30,31]. Through the use of fresh rat liver micro-some preparations, Airoldi et al. [32] and Hecht [33] were able to demonstratethat N-nitroso-2-hydroxymorpholine (NHMOR), the cyclic hemiacetal of N-nitroso-N-2-hydroxyethylethanal (3), is an oxidation product of NDELA.NHMOR is also produced by the alcohol dehydrogenase (ADH)-mediated oxida-tion of NDELA [34,35] and is further converted to NHEG (Scheme 2). ADH,in place of the normal microsomal system, is capable of inducing a mutagenicresponse from NDELA in the Ames system [34]. In their work with high-specific-activity 14C-labeled NDELA, Farrely and Lijinsky were able to produce evidencefor the formation of O6-hydroxyethylguanine (O6-HEG) and N7-hydroxyethylgu-anine (N7-HEG) adducts in DNA at low levels (Structure 2) [26]. Although thelatter adducts could be expected to arise from intermediates derived from the α-hydroxylation of NDELA, these data are the only indication that NDELA mightbe activated to some extent by α-hydroxylation.

Considerable attention has been given to the hypothesis that the β-oxidation

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178 LOEPPKY

STRUCTURE 2

of NDELA is a significant activating step. This hypothesis is principally derivedfrom three sets of observations:

1. As noted above, the only known metabolites of NDELA (Scheme 2) arederived from β-oxidation [30].

2. α-Nitrosamino aldehydes (4) (Scheme 3) such as NHMOR, derived fromthe β-oxidation of ethanolnitrosamines, are highly reactive compoundscapable of inducing nucleotide and DNA damage without further activa-tion [35–38].

3. α-Nitrosamino aldehydes are direct-acting mutagens [34,38–40]. α-Ni-trosamino aldehydes possess several unusual chemical properties. Theytransfer their N-nitroso group to other amines with ease (Scheme 3) and,either concomitant with this process or by chemistry yet to be elucidated,produce glyoxal equivalents from the two carbon chain [35–37].

Thus, NHMOR has been shown to be capable of deaminating the primary aminogroups in DNA through nitroso transfer reactions (Scheme 4). Time course andconcentration studies employing NHMOR or the more reactive butylethanalni-trosamine in reaction with calf-thymus DNA show that these transformationscould occur in vivo, although DNA base deamination is very difficult to detecttherein [37].

N-Nitroso-N-2-hydroxyethylglycine and other α-nitrosamino aldehydes alsoreact with deoxguanosine, guanine containing oligonucleotides, and DNA to form

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MECHANISM OF BIOACTIVATION OF NDELA 179

SCHEME 4

glyoxal-guanine (gG) adducts (Scheme 5) [35,37,38,41]. This fact, coupled withthe ability of the compounds to deaminate the primary amine bases in DNA,could explain the direct mutagenicity of this group of compounds. To determinewhether NDELA and related ethanolnitrosamines are capable of producing gGDNA adducts in vivo, we have developed a 32P-postlabeling assay for the gGadduct [42]. The adduct can dissociate reversibly and its stability is a functionof pH, requiring special care during the analyses. NDELA, NHMOR, methyletha-nolnitrosamine, and ethylethanolnitrosamine induce the formation of the gG ad-duct in rat liver DNA following in vivo administration of the nitrosamine. Theamount of gG adduct formed from NDELA is more than twice that arising fromNHMOR.

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Despite these experiments and others which support the hypothesis thatNDELA is activated through β-oxidation, there are several significant observa-tions which raise questions about this hypothesis. Most importantly, NHMORhas been found not to be carcinogenic in rats or AJ mice when administeredorally [43]. Although the dosages used in these experiments were rather low,parallel experiments showed NDELA to be carcinogenic to rats. More recentexperiments using two methods of analysis have shown NDELA to produce O6-hydroxyethylguanine (O6-HEG) adducts in rats in a dose-dependent manner[40,44]. No O6-HEG adducts were produced when NHMOR was administeredto rats [44]. The mutagenicity of NHMOR is significantly enhanced when it isincubated with microsomes, suggesting the role of additional CYP-mediated me-tabolism in its activation [39]. Additionally, as mentioned earlier, NHMOR pro-duces lower levels of gG adducts in DNA than NDELA [42]. These findingshave led us to step back and perform some relatively fundamental experimentsto gain new insight into the mode or modes of NDELA bioactivation.

III. REINVESTIGATION OF THE a-OXIDATION PATHWAYFOR NDELA

To reinvestigate the existence of a possible α-oxidation pathway for NDELAand to probe the relative importance of processes which involve the breaking ofeither the α- or β-CH bonds, we have prepared a set of deuterated isotopomersof NDELA and NHMOR and examined their activity toward the induction ofDNA single strand breaks (SSB) in rat liver in vivo, as measured by alkalineelution [45]. A typical result is shown in Fig. 1, where it can be seen that α-deuteration of NDELA significantly reduces the level of DNA SSB, whereas theeffect of β-D4NDELA cannot be distinguished from NDELA itself. Although theextent of the isotope effect increases with decreasing dose (1.37–3.22 for α-D4NDELA over a dose range of 0.75–0.08 mmol/kg body weight and 0.79–1.38for β-D4NDELA over the same dose range) and a small positive isotope effectfor β-D4NDELA is observed at the lowest doses, the much greater inhibition ofDNA SSB by α-D4NDELA is observed at all doses. These experiments suggestthat a process which breaks the α-CH bond is involved in the bioactivation ofNDELA.

Further evidence for this supposition was obtained in cytotoxicity experimentsperformed by Janzowski et al. [46]. The cytotoxicity of NDELA and other nitro-samines in Chinese hamster cells transfected with either CYP2E1 or CYP2B1was compared with control cells. NDELA was not cytotoxic to control cells orthose transfected with CYP2B1, whereas a significant dose-dependent cytotoxic-ity was observed for cells transfected with CYP2E1. The cytotoxicity induced by

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MECHANISM OF BIOACTIVATION OF NDELA 181

FIG. 1. Alkaline elution profiles for rat liver DNA following administrationof the indicated nitrosamine at a dose of 0.37 mM/kg body weight. The slopesof the regression lines shown in (B) were used to determine the isotope effectsof 1.88 for α-D4NDELA and 1.09 for β-D4NDELA.

NDELA and its deuterated isotopomers in CYP2E1 transfected cells was probedfurther. Cytotoxicity was observed for NDELA and β-D4NDELA but was notseen at all for α-D4NDELA (see Fig. 2) [45]. These data lent further support tothe idea that NDELA is being activated by α-hydroxylation and that NDELA isa substrate for CYP2E1.

With this evidence in hand, we have reinvestigated the work of Farrelly etal. and others which led to the conclusions that NDELA was not subject toα-hydroxylation [26,29]. As shown in Scheme 1, the decomposition of an α-hydroxynitrosamine results in the formation of a reactive diazonium ion and analdehyde or a ketone (from the α-hydroxylated chain). Assays for α-hydroxyla-tion, and indeed microsome-mediated nitrosamine metabolism, have commonlyemployed procedures which trap the aldehyde or ketone coproduct of the diazo-nium ion through derivatization and extraction into organic solvents, followedby chromatographic separation and detection in comparison with standards. Theα-hydroxylation of NDELA will lead to the formation of glycol aldehyde (8) andthe 2-hydroxyethyldiazonium ion (7), which decomposes as shown in Scheme 6.The detection of glycolaldehyde by the methodology employed by Farrely et al.[29], which works well for simple aldehydes, had to be modified considerably.Upon reaction with arylhydrazines (9), glycol aldehyde undergoes the osazonereaction shown in Scheme 7, which leads to the glyoxal bis-hydrazone (11). Be-cause glyoxal is also a possible product of metabolism, we developed extractionand derivatization procedures which permitted their independent detection as 2,4-dinitrophenylhydrazones (10 and 11) by high-performance liquid chromatogra-

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182 LOEPPKY

FIG. 2. A comparison of the cytotoxicity of NDELA (■), α-D4NDELA (d),and β-D4NDELA (m) in V79 cells transfected with CYP2E1.

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MECHANISM OF BIOACTIVATION OF NDELA 183

SCHEME 7

phy (HPLC) [47]. This methodology required the much more polar extractionsolvent CH2Cl2, compared to isooctane used by Farelly et al. [29].

Through the synthesis and decomposition of α-acetoxy-NDELA (6), we dem-onstrated that the intermediate α-hydroxynitrosamine (5) did indeed generate gly-col aldehyde (8) and acetaldehyde as shown in Scheme 6 [48], but because acetal-dehyde is produced by other metabolic processes, glycol aldehyde productionwas utilized as a monitor of NDELA α-oxidation. Rat liver microsomes fromanimals which had been preinduced with different drugs were utilized. Glycolaldehyde could be detected in each case [47]. NHMOR was formed through β-oxidation in even larger amounts, and glyoxal was also a product. The metaboliccapacity of various microsomes was compared with their ability to oxidize di-methylnitrosamine to formaldehyde, a process known to be catalyzed byCYP2E1, and the data are presented in Fig. 3. It is obvious at once that the β-oxidation of NDELA to give NHMOR shows the same profile with the differentmicrosome preparations, as is exhibited by dimethylnitrosamine (DMN) α-hydroxylation, suggesting similar enzyme selectivity for both substrates. A simi-lar profile is observed for the formation of glycol aldehyde from NDELA. Bothof the most effective microsomal preparations, those induced by either isoniazidor streptozotocine, are known to be rich in CYP2E1. The failure of previousworkers to detect microsome-mediated α-oxidation of NDELA was probably theresult of two significant factors: inadequate analytical methodology and the lackof good microsomal systems. NDELA is a relatively poor substrate and consider-ably more is known now about the generation of microsomal preparations highin CYP2E1 than was known during the time of the previous work. Figure 3 showsthat microsomes from uninduced animals are inefficient in NDELA metabolism.Moreover, a much lower concentration of DMN than NDELA was required togenerate the levels of formaldehyde shown in Fig. 3 because it is a much bettersubstrate.

Additional experiments utilizing isoniazid-induced microsomes reveal severalinteresting phenomena (see Scheme 8). As can be seen from inspection of Table1, the microsomal oxidation of NDELA results in approximately twice as much

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FIG. 3. The effect of preinduction of animals with various drugs on thecapacity of rat liver microsomes to metabolize either dimethylnitrosamine (DMN)(1 mM ) or NDELA (20 mM ) is shown. The similar profiles strongly suggest thatNDELA is most effectively metabolized by CYP2E1, the isoenzyme known tobe most effective in the metabolism of DMN. (Preinducers: STR 5 streptazoicin;ISO 5 isoniazid; PB 5 phenobarbital; N 5 no preinduction).

β-oxidation as α-oxidation [47]. CYP2E1 is known to catalyze the oxidation ofethanol to acetaldehyde and to act on hydrophilic substrates. We also determinedthat glycol aldehyde (8) is oxidized to glyoxal (12) by this microsomal system.This observation was somewhat of a surprise, because all of the literature suggeststhat glycol aldehyde, an intermediate in the metabolism of ethylene glycol, isoxidized to glycolic acid and then further to oxalic acid. The latter process maybe a faster route, but our data would suggest that glyoxal must be considered apossible toxic metabolite of ethylene glycol. NHMOR is oxidized further to both

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MECHANISM OF BIOACTIVATION OF NDELA 185

TABLE 1

Rates of Product Formation from the Isoniazid-Induced MicrosomalOxidation of NDELA and Related Metabolites

Product (nmol/mg prot./min)

Substrate Glycol aldehyde NHMOR Glyoxal

NDELA 20 mM 2.4 5 0.2Glycol aldehyde 0.4 mM — — 1.36NHMOR 0.4 mM 0.58 — 0.26

glyoxal and glycol aldehyde. α-Oxidation of this unsymmetrical nitrosamine ateither of the carbons adjacent to the N could produce these products, with glyoxalarising from α-hydroxylation of the more oxidized side chain.

The microsomal oxidation of NDELA, α-D4NDELA, and β-D4NDELA werecompared [47]. As is evident from Fig. 4, deuterium substitution results in sig-nificant switching of the metabolic profile. α-Deuteration practically eliminatesα-oxidation and glycol aldehyde formation. β-Deuteration reduces NHMOR for-mation by half and doubles the glycol aldehyde formation. Glyoxal formation,

FIG. 4. The effect of deuteration at either of the two carbons of NDELAon the metabolic selectivity of isoniazid-induced rat liver microsomes is shown.

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186 LOEPPKY

which arises from a second oxidative step and requires both α- and β-oxidation,is reduced by both α- and β-deuteration.

The near elimination of glycol aldehyde formation by α-deuteration ofNDELA agrees well with both the deuterium isotope effect on CYP2E1transfected cell cytotoxicity and the decreased DNA SSB observed for this iso-topomer. In order to further test the connection between these phenomena, wesought to determine whether we could observe a deuterium isotope effect onDNA adduct formation from NDELA. 32P-Postlabeling assays of DNA are amongthe most sensitive methods and can be specific when utilized with well-character-ized standards. Toward this end, we have prepared O6-2-hydroxyethyldeoxygua-nidine-5′-32P-phosphate as a standard [49,50] and used it in a 32P-postlabelingassay of the same rat liver DNA used in the DNA SSB experiments. The 32P-postlabeling assay involves the enzymatic digestion of DNA to 3′-nucleotidephosphates, a HPLC cleanup step to remove significant amounts of the unmodi-fied nucleotides, enzymatic labeling at the 5′-position with 32P-ATP, enzymaticcleavage of the 3′-phosphate, and HPLC radiometric detection along with sepa-rate two-dimensional thin-layer chromatography separations followed by autora-diography. Using this sensitive methodology with the standard, we were able todemonstrate the formation of HEG adducts in the liver DNA of NDELA-treatedrats at all doses examined. Moreover, although 32P-postlabeling data are difficultto quantitate, deuterium isotope effects could clearly be seen for the formationof HEG adducts, as is shown in Table 2. Although there was some variation fromanimal to animal and from one 32P-postlabeling run to another, in every caseHEG adduct levels in the DNA of rats treated with NDELA (0.75 mmol/kg bodyweight) were greater than those observed in animals treated with α-D4NDELA,and HEG adduct levels for β-D4NDELA were significantly larger than those ob-served for NDELA. These data agree well with the data obtained for the micro-somal metabolism of NDELA and its deuterated isotopomers. α-Deuteration re-duces α-hydroxylation and the formation of the 2-hydroxyethyl diazonium ionwhich gives rise to the HEG adduct. In the microsomal metabolism experiments,

TABLE 2

The Effect of Deuterium Substitution on HEGDNA Adduct Levels

Substrate Adduct levela Isotope effect

NDELA 0.026 —α-D4NDELA 0.007 3.7β-D4NDELA 0.100 0.26

a ng adduct/10 µg DNA.

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MECHANISM OF BIOACTIVATION OF NDELA 187

this effect was measured through the formation of the other product, glycol alde-hyde. On the other hand, β-deuteration enhances α-hydroxylation through meta-bolic switching and greater amounts of HEG adducts are seen for β-D4NDELA.Whereas the effect of deuterium substitution on HEG DNA adduct formationagrees well with the results of our microsomal metabolism experiments, the en-hancement in HEG adduct formation for β-D4NDELA does not show the samestrong correlation with isotope effects on DNA SSB as is observed for α-D4NDELA. A modest inverse isotope effect (0.8) was observed on DNA SSBfor β-D4NDELA at the highest dose level, indicating that strand breaks are in-creased by β-D4NDELA; however, at the lowest dose of β-D4NDELA, the isotopeeffect is 1.4, showing at this dose level DNA SSB is inhibited by β-deuteration.Clarification of these effects should be forthcoming when the isotope effects ongG adduct formation are determined. The lower stability of this adduct due toits pH-dependent reversible formation makes determinations of isotope effectson its formation more difficult.

The data described above are clearly indicative of the existence of an α-oxida-tion pathway for NDELA. α-Deuteration reduces α-hydroxylation of NDELAby means of a primary isotope effect. The results of this are a decrease in thelevel of diazonium formation and a corresponding decrease in the level of DNAalkylation. We have demonstrated that α-deuteration reduces the level of O6-HEG adducts. Diazonium ions alkylate DNA bases at sites other than just O6 ofguanine. Alkylation on the purine imidazole nitrogens (primarily N7) and at N1

or N3 of adenine can result in depurination and the generation of abasic sites.Repair of these sites and other lesions is one mechanism for an increase in DNASSB. A decrease in the number of these lesions through the α-deuterium isotopeeffect will decrease the level of DNA SSB. The fact that the cytotoxicity ofNDELA is eliminated by α-deuteration in CYP2E1 transfected cells indicatesthat this isoenzyme is involved in NDELA activation to cytotoxic metabolites aswell.

IV. OTHER BIOACTIVATION PATHWAYS

The fact that NDELA also forms gG adducts in vivo, a process which requiresboth α- and β-oxidation, and the observation that microsomes are more efficientin the oxidation of NDELA to NHMOR than their catalysis of α-oxidation (seeTable 1, entry 1) suggest that the mechanism of NDELA activation involves morethan just α-hydroxylation. It is obvious from the results described above thatglycol aldehyde could be further oxidized to glyoxal, which could react withDNA. α-Hydroxylation of NHMOR to (16) (Scheme 9) would lead to the forma-tion of both glyoxal (12) and the 2-hydroxyethyldiazonium ion (7). We have

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188 LOEPPKY

SCHEME 9

modeled this chemistry through the hydrolysis of 2,3-epoxy-N-nitrosomorpholine(15) and demonstrated both decomposition products and adducts expected of α-hydroxy-NHMOR (e.g., gG and O6-HEG adducts). The only problem with thispathway, α-hydroxylation of NHMOR, lies with the lack of carcinogenicity ofNHMOR [43]. In the carcinogenicity experiments, low concentrations ofNHMOR were given in drinking water. Introduction of this relatively water-solu-ble compound by this method may result in its exclusion from the cell compart-ments in which it is generated by metabolic oxidation of NDELA.

Several other hypotheses for the activation of NDELA and related ethanolnitrosamines require discussion. Some years ago, Michejda and colleagues dem-onstrated that the tosylate ester (17) of methylethanolnitrosamine (19) cyclizedto 3-methyl-1,2,3-oxadiazolinium tosylate (18) on mild heating (Scheme 10) [51].These researchers proposed this process as an organic chemical model for trans-formations of ethanol nitrosamines, which may follow their being conjugated

SCHEME 10

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MECHANISM OF BIOACTIVATION OF NDELA 189

with sulfate [e.g., (19) → (20), Scheme 10] [52–56]. Sulfate esters of β-hy-droxynitrosamines, which are secondary alcohols, have been isolated as urinarymetabolites. It was proposed that 3-methyl-1,2,3-oxadiazolinium ions acted invivo by methylating and hydroxyethylating DNA. Although several publicationspresent data and discussions supporting this hypothesis, other experimental dataraise serious questions regarding the existence of this pathway for methylethanol-nitrosamine, and little data relative to NDELA activation by this pathway haveappeared.

Methylethanolnitrosamine is known to produce methyl- and 2-hydroxyethyl-DNA adducts in vivo. This adduction process is inhibited by 2-propanol, a knownsulfotransferase competitive inhibitor [52,53,56]. 2-Propanol, however, is not aspecific inhibitor and also inhibits ADH and microsomal oxidation of NDELA,for example. The DNA alkylation characteristics of a strain of brachymorphicmice deficient in PAPS, a sulfotransferase cofactor, did not differ from controlswhen treated with methylethanolnitrosamine [52,53]. We have demonstrated that3-methyl-1,2,3-oxadiazolinium tosylate does not 2-hydroxyethylate DNA orother guanine derivatives [49]. Its major reaction (Scheme 11) with guanine isto introduce the entire nitrosamine fragment at N7 to produce (21). This processoccurs to a much greater extent than methylation. Moreover, we have shown thatmethyl-1,2,3-oxadiazolinium tosylate oxidizes thiols very rapidly [57]. Whereasthis process involves the probable generation of free radicals, it occurs so rapidlyas to likely prevent the alkylation of DNA by 1,2,3-oxadiazolinium ions, ifformed. Taken together with the conflicting biological data, it seems unlikelythat activation through this mode of sulfation plays a role in the carcinogenicaction of ethanol nitrosamines.

Sterzel et al. observed a decrease in DNA SSB induced by NDELA whenthe sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol was administered in 2-propanol [58,59]. The latter solvent was not realized to be an enzyme inhibitor.DNA SSB induced by NDELA are also inhibited by butylthiolane-S-oxide, whichis purported to be an ADH inhibitor. On the basis of these data, it was suggestedthat NDELA was activated through both ADH and sulfotransferase-mediated pro-cesses. The first step was conceived to involve β-oxidation of NDELA toNHMOR, which was perceived to be followed by sulfation of the OH group of

SCHEME 11

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the hemiacetal to generate a reactive electrophile. Because of the neighboringoxygen atom, this sulfate ester can reasonably be anticipated to be reactive. Wehave prepared the corresponding tosylate (13) (Scheme 9), and under the condi-tions of our experiment shown, it rapidly undergoes elimination to produce 2,3-dehydro-N-nitrosomorpholine (14) [60]. Should this transformation occur in vivo,it could explain activation by processes which break, at different times, both theα- and β-CH bonds of NDELA. The epoxidation of 14 by CYP, should it occur,leads to the highly reactive epoxide (15), the chemistry of which is discussedabove and does produce both gG and O6-HEG adducts in vitro. But this processinvolves the breaking of the α-CH bond in an elimination step, which is notlikely to be catalyzed by CYP2E1. Although the activation of NDELA by thelatter pathway is supported by model chemistry, additional supporting data mustbe acquired before it can be considered seriously.

V. CONCLUSION

Here, we have summarized and analyzed the experimental data supportingvarious activation pathways for NDELA. We now have strong evidence thatNDELA is activated in part by α-hydroxylation. The discovery of the existenceof this pathway for NDELA activation in early experiments was impeded by thelack of an adequate assay for α-hydroxylation products and the lack of properlyinduced microsomal systems. It is clear that another oxidation is required in orderto generate the gG adduct. Although we have generated some relevant informa-tion on how this could occur, more work will have to be done to elucidate thein vivo pathway to this adduct.

ACKNOWLEDGMENTS

The support of this research by a grant from the National Institute of Environ-mental Health Sciences (RO1 ES 03953) and a Senior Research Fellowship fromthe Fogarty Foundation (NIH FO6 TW01944) is gratefully acknowledged.

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