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DRUG METABOLISM REVIEWS, 31(1), 175193 (1999)

THE MECHANISM OF BIOACTIVATION

OF N-NITROSODIETHANOLAMINE*
RICHARD N. LOEPPKY
Department of Chemistry
University of Missouri
Columbia, Missouri 65211

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

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

III. REINVESTIGATION OF THE -OXIDATION PATHWAY FOR

NDELA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 widespread 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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STRUCTURE 1

nolamine, triethanolamine, and various derivatives thereof which are used in a

great many formulations ranging from cosmetics and personal care items to metalworking fluids [19]. Despite the fact that it has been demonstrated to be a
potent carcinogen in several species of animals by means of dose-response studies
[1013], its mode of carcinogenic activation has remained relatively obscure.
The purpose of this review is to summarize various hypotheses related to its
mode of bioactivation and how new data discriminate between them and point
the direction of further investigation.
Most nitrosamines are believed to be activated by cytochrome P450 (CYP)mediated -hydroxylation as depicted in Scheme 1 [14,15]. -Hydroxynitrosamines (1) have been synthesized and shown to decompose rapidly in aqueous solution with evolution of N 2, which is assumed to arise from a diazonium ion intermediate (2) [16,17]. -Acetoxynitrosamines are often used as relatively stable
progenitors of -hydroxynitrosamines and are helpful in testing hypotheses regarding the nature of metabolites, as well as protein and nucleotide adducts expected from metabolically generated diazonium ions [1820]. Numerous studies
have revealed a similarity between in vivo nitrosamine-produced alkylation products and those derived from -acetoxynitrosamines in vitro and in vivo, thereby
strengthening the view that the bioactivation of many nitrosamines involves hydroxylation [21,22].

SCHEME 1

MECHANISM OF BIOACTIVATION OF NDELA

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II. IS NDELA ACTIVATED BY -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 is
excreted in the urine unchanged, even at low doses [2325]. Compared to other
nitrosamines, NDELA results in very low levels of 14 C incorporation into DNA
[23,26]. NDELA is not mutagenic in the standard microsome-mediated Ames
mutagenicity assay [27,28]. Other studies have failed to observe products expected of the metabolic -hydroxylation of NDELA using either microsomal
preparations or primary rat hepatocytes [29]. The latter findings have been used
to explain the failure of NDELA to produce a mutagenic response in microsomemediated assays.
The metabolic disposition of NDELA (Scheme 2) has been investigated by
Airoldi and co-workers, who found a single urinary metabolite, N-nitroso-N-2hydroxyethylgycine (NHEG) [30,31]. Through the use of fresh rat liver microsome preparations, Airoldi et al. [32] and Hecht [33] were able to demonstrate
that N-nitroso-2-hydroxymorpholine (NHMOR), the cyclic hemiacetal of Nnitroso-N-2-hydroxyethylethanal (3), is an oxidation product of NDELA.
NHMOR is also produced by the alcohol dehydrogenase (ADH)-mediated oxidation 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 mutagenic
response from NDELA in the Ames system [34]. In their work with high-specificactivity 14 C-labeled NDELA, Farrely and Lijinsky were able to produce evidence
for the formation of O 6-hydroxyethylguanine (O 6-HEG) and N 7-hydroxyethylguanine (N 7-HEG) adducts in DNA at low levels (Structure 2) [26]. Although the
latter adducts could be expected to arise from intermediates derived from the hydroxylation of NDELA, these data are the only indication that NDELA might
be activated to some extent by -hydroxylation.
Considerable attention has been given to the hypothesis that the -oxidation

SCHEME 2

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STRUCTURE 2

of NDELA is a significant activating step. This hypothesis is principally derived

from three sets of observations:
1. As noted above, the only known metabolites of NDELA (Scheme 2) are
derived from -oxidation [30].
2. -Nitrosamino aldehydes (4) (Scheme 3) such as NHMOR, derived from
the -oxidation of ethanolnitrosamines, are highly reactive compounds
capable of inducing nucleotide and DNA damage without further activation [3538].
3. -Nitrosamino aldehydes are direct-acting mutagens [34,3840]. -Nitrosamino aldehydes possess several unusual chemical properties. They
transfer 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 [3537].
Thus, NHMOR has been shown to be capable of deaminating the primary amino
groups in DNA through nitroso transfer reactions (Scheme 4). Time course and
concentration studies employing NHMOR or the more reactive butylethanalnitrosamine in reaction with calf-thymus DNA show that these transformations
could occur in vivo, although DNA base deamination is very difficult to detect
therein [37].
N-Nitroso-N-2-hydroxyethylglycine and other -nitrosamino aldehydes also
react with deoxguanosine, guanine containing oligonucleotides, and DNA to form

SCHEME 3

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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 with

the ability of the compounds to deaminate the primary amine bases in DNA,
could explain the direct mutagenicity of this group of compounds. To determine
whether NDELA and related ethanolnitrosamines are capable of producing gG
DNA adducts in vivo, we have developed a 32 P-postlabeling assay for the gG
adduct [42]. The adduct can dissociate reversibly and its stability is a function
of pH, requiring special care during the analyses. NDELA, NHMOR, methylethanolnitrosamine, and ethylethanolnitrosamine induce the formation of the gG adduct in rat liver DNA following in vivo administration of the nitrosamine. The
amount of gG adduct formed from NDELA is more than twice that arising from
NHMOR.

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Despite these experiments and others which support the hypothesis that
NDELA is activated through -oxidation, there are several significant observations which raise questions about this hypothesis. Most importantly, NHMOR
has been found not to be carcinogenic in rats or AJ mice when administered
orally [43]. Although the dosages used in these experiments were rather low,
parallel experiments showed NDELA to be carcinogenic to rats. More recent
experiments using two methods of analysis have shown NDELA to produce O 6hydroxyethylguanine (O 6-HEG) adducts in rats in a dose-dependent manner
[40,44]. No O 6-HEG adducts were produced when NHMOR was administered
to rats [44]. The mutagenicity of NHMOR is significantly enhanced when it is
incubated with microsomes, suggesting the role of additional CYP-mediated metabolism in its activation [39]. Additionally, as mentioned earlier, NHMOR produces lower levels of gG adducts in DNA than NDELA [42]. These findings
have led us to step back and perform some relatively fundamental experiments
to gain new insight into the mode or modes of NDELA bioactivation.

III. REINVESTIGATION OF THE -OXIDATION PATHWAY

FOR NDELA
To reinvestigate the existence of a possible -oxidation pathway for NDELA
and to probe the relative importance of processes which involve the breaking of
either the - or -CH bonds, we have prepared a set of deuterated isotopomers
of NDELA and NHMOR and examined their activity toward the induction of
DNA single strand breaks (SSB) in rat liver in vivo, as measured by alkaline
elution [45]. A typical result is shown in Fig. 1, where it can be seen that deuteration of NDELA significantly reduces the level of DNA SSB, whereas the
effect of -D 4NDELA cannot be distinguished from NDELA itself. Although the
extent of the isotope effect increases with decreasing dose (1.373.22 for D 4NDELA over a dose range of 0.750.08 mmol/kg body weight and 0.791.38
for -D 4NDELA over the same dose range) and a small positive isotope effect
for -D 4NDELA is observed at the lowest doses, the much greater inhibition of
DNA SSB by -D 4NDELA is observed at all doses. These experiments suggest
that a process which breaks the -CH bond is involved in the bioactivation of
NDELA.
Further evidence for this supposition was obtained in cytotoxicity experiments
performed by Janzowski et al. [46]. The cytotoxicity of NDELA and other nitrosamines in Chinese hamster cells transfected with either CYP2E1 or CYP2B1
was compared with control cells. NDELA was not cytotoxic to control cells or
those transfected with CYP2B1, whereas a significant dose-dependent cytotoxicity 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 administration
of the indicated nitrosamine at a dose of 0.37 mM/kg body weight. The slopes
of the regression lines shown in (B) were used to determine the isotope effects
of 1.88 for -D 4NDELA and 1.09 for -D 4NDELA.
NDELA and its deuterated isotopomers in CYP2E1 transfected cells was probed
further. Cytotoxicity was observed for NDELA and -D 4NDELA but was not
seen at all for -D 4NDELA (see Fig. 2) [45]. These data lent further support to
the idea that NDELA is being activated by -hydroxylation and that NDELA is
a substrate for CYP2E1.
With this evidence in hand, we have reinvestigated the work of Farrelly et
al. and others which led to the conclusions that NDELA was not subject to
-hydroxylation [26,29]. As shown in Scheme 1, the decomposition of an hydroxynitrosamine results in the formation of a reactive diazonium ion and an
aldehyde or a ketone (from the -hydroxylated chain). Assays for -hydroxylation, and indeed microsome-mediated nitrosamine metabolism, have commonly
employed procedures which trap the aldehyde or ketone coproduct of the diazonium ion through derivatization and extraction into organic solvents, followed
by chromatographic separation and detection in comparison with standards. The
-hydroxylation of NDELA will lead to the formation of glycol aldehyde (8) and
the 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 osazone
reaction shown in Scheme 7, which leads to the glyoxal bis-hydrazone (11). Because glyoxal is also a possible product of metabolism, we developed extraction
and derivatization procedures which permitted their independent detection as 2,4dinitrophenylhydrazones (10 and 11) by high-performance liquid chromatogra-

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FIG. 2. A comparison of the cytotoxicity of NDELA (), -D 4 NDELA (),

and -D 4 NDELA () 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 extraction
solvent CH 2Cl 2, compared to isooctane used by Farelly et al. [29].
Through the synthesis and decomposition of -acetoxy-NDELA (6), we demonstrated that the intermediate -hydroxynitrosamine (5) did indeed generate glycol aldehyde (8) and acetaldehyde as shown in Scheme 6 [48], but because acetaldehyde is produced by other metabolic processes, glycol aldehyde production
was utilized as a monitor of NDELA -oxidation. Rat liver microsomes from
animals which had been preinduced with different drugs were utilized. Glycol
aldehyde could be detected in each case [47]. NHMOR was formed through oxidation in even larger amounts, and glyoxal was also a product. The metabolic
capacity of various microsomes was compared with their ability to oxidize dimethylnitrosamine to formaldehyde, a process known to be catalyzed by
CYP2E1, and the data are presented in Fig. 3. It is obvious at once that the oxidation of NDELA to give NHMOR shows the same profile with the different
microsome preparations, as is exhibited by dimethylnitrosamine (DMN) hydroxylation, suggesting similar enzyme selectivity for both substrates. A similar profile is observed for the formation of glycol aldehyde from NDELA. Both
of the most effective microsomal preparations, those induced by either isoniazid
or streptozotocine, are known to be rich in CYP2E1. The failure of previous
workers to detect microsome-mediated -oxidation of NDELA was probably the
result of two significant factors: inadequate analytical methodology and the lack
of good microsomal systems. NDELA is a relatively poor substrate and considerably more is known now about the generation of microsomal preparations high
in CYP2E1 than was known during the time of the previous work. Figure 3 shows
that microsomes from uninduced animals are inefficient in NDELA metabolism.
Moreover, a much lower concentration of DMN than NDELA was required to
generate the levels of formaldehyde shown in Fig. 3 because it is a much better
substrate.
Additional experiments utilizing isoniazid-induced microsomes reveal several
interesting phenomena (see Scheme 8). As can be seen from inspection of Table
1, the microsomal oxidation of NDELA results in approximately twice as much

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LOEPPKY

FIG. 3. The effect of preinduction of animals with various drugs on the

capacity of rat liver microsomes to metabolize either dimethylnitrosamine (DMN)
(1 mM ) or NDELA (20 mM ) is shown. The similar profiles strongly suggest that
NDELA is most effectively metabolized by CYP2E1, the isoenzyme known to
be most effective in the metabolism of DMN. (Preinducers: STR streptazoicin;
ISO isoniazid; PB phenobarbital; N no preinduction).
-oxidation as -oxidation [47]. CYP2E1 is known to catalyze the oxidation of
ethanol to acetaldehyde and to act on hydrophilic substrates. We also determined
that glycol aldehyde (8) is oxidized to glyoxal (12) by this microsomal system.
This observation was somewhat of a surprise, because all of the literature suggests
that glycol aldehyde, an intermediate in the metabolism of ethylene glycol, is
oxidized to glycolic acid and then further to oxalic acid. The latter process may
be a faster route, but our data would suggest that glyoxal must be considered a
possible toxic metabolite of ethylene glycol. NHMOR is oxidized further to both

SCHEME 8

MECHANISM OF BIOACTIVATION OF NDELA

185

TABLE 1
Rates of Product Formation from the Isoniazid-Induced Microsomal
Oxidation of NDELA and Related Metabolites

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Product (nmol/mg prot./min)

Substrate
NDELA 20 mM
Glycol aldehyde 0.4 mM
NHMOR 0.4 mM

Glycol aldehyde

NHMOR

Glyoxal

2.4

0.58

0.2
1.36
0.26

glyoxal and glycol aldehyde. -Oxidation of this unsymmetrical nitrosamine at

either of the carbons adjacent to the N could produce these products, with glyoxal
arising from -hydroxylation of the more oxidized side chain.
The microsomal oxidation of NDELA, -D 4NDELA, and -D 4NDELA were
compared [47]. As is evident from Fig. 4, deuterium substitution results in significant switching of the metabolic profile. -Deuteration practically eliminates
-oxidation and glycol aldehyde formation. -Deuteration reduces NHMOR formation by half and doubles the glycol aldehyde formation. Glyoxal formation,

FIG. 4. The effect of deuteration at either of the two carbons of NDELA

on the metabolic selectivity of isoniazid-induced rat liver microsomes is shown.

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LOEPPKY

which arises from a second oxidative step and requires both - and -oxidation,
is reduced by both - and -deuteration.
The near elimination of glycol aldehyde formation by -deuteration of
NDELA agrees well with both the deuterium isotope effect on CYP2E1
transfected cell cytotoxicity and the decreased DNA SSB observed for this isotopomer. In order to further test the connection between these phenomena, we
sought to determine whether we could observe a deuterium isotope effect on
DNA adduct formation from NDELA. 32 P-Postlabeling assays of DNA are among
the most sensitive methods and can be specific when utilized with well-characterized standards. Toward this end, we have prepared O 6-2-hydroxyethyldeoxyguanidine-5-32 P-phosphate as a standard [49,50] and used it in a 32 P-postlabeling
assay of the same rat liver DNA used in the DNA SSB experiments. The 32 Ppostlabeling assay involves the enzymatic digestion of DNA to 3-nucleotide
phosphates, a HPLC cleanup step to remove significant amounts of the unmodified nucleotides, enzymatic labeling at the 5-position with 32 P-ATP, enzymatic
cleavage of the 3-phosphate, and HPLC radiometric detection along with separate two-dimensional thin-layer chromatography separations followed by autoradiography. Using this sensitive methodology with the standard, we were able to
demonstrate the formation of HEG adducts in the liver DNA of NDELA-treated
rats at all doses examined. Moreover, although 32 P-postlabeling data are difficult
to quantitate, deuterium isotope effects could clearly be seen for the formation
of HEG adducts, as is shown in Table 2. Although there was some variation from
animal to animal and from one 32 P-postlabeling run to another, in every case
HEG adduct levels in the DNA of rats treated with NDELA (0.75 mmol/kg body
weight) were greater than those observed in animals treated with -D4NDELA,
and HEG adduct levels for -D 4NDELA were significantly larger than those observed for NDELA. These data agree well with the data obtained for the microsomal metabolism of NDELA and its deuterated isotopomers. -Deuteration reduces -hydroxylation and the formation of the 2-hydroxyethyl diazonium ion
which gives rise to the HEG adduct. In the microsomal metabolism experiments,

TABLE 2
The Effect of Deuterium Substitution on HEG
DNA Adduct Levels
Substrate
NDELA
-D 4NDELA
-D 4NDELA
a

Adduct level a

Isotope effect

0.026
0.007
0.100

3.7
0.26

ng adduct/10 g DNA.

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MECHANISM OF BIOACTIVATION OF NDELA

187

this effect was measured through the formation of the other product, glycol aldehyde. On the other hand, -deuteration enhances -hydroxylation through metabolic switching and greater amounts of HEG adducts are seen for -D 4NDELA.
Whereas the effect of deuterium substitution on HEG DNA adduct formation
agrees well with the results of our microsomal metabolism experiments, the enhancement in HEG adduct formation for -D 4NDELA does not show the same
strong correlation with isotope effects on DNA SSB as is observed for D 4NDELA. A modest inverse isotope effect (0.8) was observed on DNA SSB
for -D 4NDELA at the highest dose level, indicating that strand breaks are increased by -D 4NDELA; however, at the lowest dose of -D 4NDELA, the isotope
effect is 1.4, showing at this dose level DNA SSB is inhibited by -deuteration.
Clarification of these effects should be forthcoming when the isotope effects on
gG adduct formation are determined. The lower stability of this adduct due to
its pH-dependent reversible formation makes determinations of isotope effects
on its formation more difficult.
The data described above are clearly indicative of the existence of an -oxidation pathway for NDELA. -Deuteration reduces -hydroxylation of NDELA
by means of a primary isotope effect. The results of this are a decrease in the
level of diazonium formation and a corresponding decrease in the level of DNA
alkylation. We have demonstrated that -deuteration reduces the level of O 6HEG adducts. Diazonium ions alkylate DNA bases at sites other than just O 6 of
guanine. Alkylation on the purine imidazole nitrogens (primarily N 7) and at N 1
or N 3 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 DNA
SSB. A decrease in the number of these lesions through the -deuterium isotope
effect will decrease the level of DNA SSB. The fact that the cytotoxicity of
NDELA is eliminated by -deuteration in CYP2E1 transfected cells indicates
that this isoenzyme is involved in NDELA activation to cytotoxic metabolites as
well.

IV.

OTHER BIOACTIVATION PATHWAYS

The fact that NDELA also forms gG adducts in vivo, a process which requires
both - and -oxidation, and the observation that microsomes are more efficient
in the oxidation of NDELA to NHMOR than their catalysis of -oxidation (see
Table 1, entry 1) suggest that the mechanism of NDELA activation involves more
than just -hydroxylation. It is obvious from the results described above that
glycol aldehyde could be further oxidized to glyoxal, which could react with
DNA. -Hydroxylation of NHMOR to (16) (Scheme 9) would lead to the formation of both glyoxal (12) and the 2-hydroxyethyldiazonium ion (7). We have

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SCHEME 9

modeled this chemistry through the hydrolysis of 2,3-epoxy-N-nitrosomorpholine

(15) and demonstrated both decomposition products and adducts expected of hydroxy-NHMOR (e.g., gG and O 6-HEG adducts). The only problem with this
pathway, -hydroxylation of NHMOR, lies with the lack of carcinogenicity of
NHMOR [43]. In the carcinogenicity experiments, low concentrations of
NHMOR were given in drinking water. Introduction of this relatively water-soluble compound by this method may result in its exclusion from the cell compartments in which it is generated by metabolic oxidation of NDELA.
Several other hypotheses for the activation of NDELA and related ethanol
nitrosamines require discussion. Some years ago, Michejda and colleagues demonstrated that the tosylate ester (17) of methylethanolnitrosamine (19) cyclized
to 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 transformations 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] [5256]. Sulfate esters of -hydroxynitrosamines, which are secondary alcohols, have been isolated as urinary
metabolites. It was proposed that 3-methyl-1,2,3-oxadiazolinium ions acted in
vivo by methylating and hydroxyethylating DNA. Although several publications
present data and discussions supporting this hypothesis, other experimental data
raise serious questions regarding the existence of this pathway for methylethanolnitrosamine, and little data relative to NDELA activation by this pathway have
appeared.
Methylethanolnitrosamine is known to produce methyl- and 2-hydroxyethylDNA adducts in vivo. This adduction process is inhibited by 2-propanol, a known
sulfotransferase competitive inhibitor [52,53,56]. 2-Propanol, however, is not a
specific inhibitor and also inhibits ADH and microsomal oxidation of NDELA,
for example. The DNA alkylation characteristics of a strain of brachymorphic
mice deficient in PAPS, a sulfotransferase cofactor, did not differ from controls
when treated with methylethanolnitrosamine [52,53]. We have demonstrated that
3-methyl-1,2,3-oxadiazolinium tosylate does not 2-hydroxyethylate DNA or
other guanine derivatives [49]. Its major reaction (Scheme 11) with guanine is
to introduce the entire nitrosamine fragment at N 7 to produce (21). This process
occurs to a much greater extent than methylation. Moreover, we have shown that
methyl-1,2,3-oxadiazolinium tosylate oxidizes thiols very rapidly [57]. Whereas
this process involves the probable generation of free radicals, it occurs so rapidly
as to likely prevent the alkylation of DNA by 1,2,3-oxadiazolinium ions, if
formed. Taken together with the conflicting biological data, it seems unlikely
that activation through this mode of sulfation plays a role in the carcinogenic
action of ethanol nitrosamines.
Sterzel et al. observed a decrease in DNA SSB induced by NDELA when
the sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol was administered in 2propanol [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, which
is purported to be an ADH inhibitor. On the basis of these data, it was suggested
that NDELA was activated through both ADH and sulfotransferase-mediated processes. The first step was conceived to involve -oxidation of NDELA to
NHMOR, which was perceived to be followed by sulfation of the OH group of

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the hemiacetal to generate a reactive electrophile. Because of the neighboring

oxygen atom, this sulfate ester can reasonably be anticipated to be reactive. We
have prepared the corresponding tosylate (13) (Scheme 9), and under the conditions of our experiment shown, it rapidly undergoes elimination to produce 2,3dehydro-N-nitrosomorpholine (14) [60]. Should this transformation occur in vivo,
it could explain activation by processes which break, at different times, both the
- and -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 discussed
above and does produce both gG and O 6-HEG adducts in vitro. But this process
involves the breaking of the -CH bond in an elimination step, which is not
likely to be catalyzed by CYP2E1. Although the activation of NDELA by the
latter pathway is supported by model chemistry, additional supporting data must
be acquired before it can be considered seriously.

V. CONCLUSION
Here, we have summarized and analyzed the experimental data supporting
various activation pathways for NDELA. We now have strong evidence that
NDELA is activated in part by -hydroxylation. The discovery of the existence
of this pathway for NDELA activation in early experiments was impeded by the
lack of an adequate assay for -hydroxylation products and the lack of properly
induced microsomal systems. It is clear that another oxidation is required in order
to generate the gG adduct. Although we have generated some relevant information on how this could occur, more work will have to be done to elucidate the
in vivo pathway to this adduct.

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

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