Lactone metabolite common to the carcinogens dioxane, diethylene glycol, and N-nitrosomorpholine: Aqueous chemistry and failure to mediate liver carcinogenesis in the F344 rat

Article abstract of DOI:10.1021/tx3000076

1,4-Dioxan-2-one, 1, was synthesized, and the equilibrium constant between it and the hydrolysis product 2-(2-hydroxyethoxy) acetic acid, 2, was determined as K_O = 70 ± 4 in acidic aqueous media, 25 °C, ionic strength 1 M (KCl), and 5% by volume acetonitrile. The carboxylic acid dissociation constant of 2 was determined under the same conditions to be pK_a = 3.31 ± 0.02. On the basis of these two determinations, the equilibrium constant between 1 and carboxylic acid anion, 3, and the proton was calculated to be K_OA = 0.034 ± 0.002 M. The stability of 1 was determined in the range of pH between 1 and 8.5 in buffered aqueous solutions under the conditions above by UV spectrophotometric methods and exhibited simple first order kinetics of decay. On the basis of buffer dilution plots, the values of k_o, the rate constant for solvent mediated decomposition, were determined. The plot of log k_o against pH is consistent with a three term rate law for solvolysis with a hydrogen ion catalyzed rate constant k_H+ = 1.1 (±0.1) M^-1 min^-1, a water catalyzed rate constant, k_w = 9.9 (±0.5) × 10^-4 min^-1, and a hydroxide ion catalyzed rate constant, k_OH = 4.1 (±0.3) × 10 ⁴ M^-1 min^-1. The t_1/2 for decay at pH 7.0, at 25 °C, is ～2 h. Treatment of F344 rats with aflatoxin B ₁ (AFB₁) (positive control) elicited the expected preneoplastic foci in the livers of each rat tested, while subsequent administration of 1 (a total of 1.32 g over 12 weeks) failed to statistically increase focal number or focal volume percent. In 8 rats administered 1 (1.32 g, 12 weeks) alone, no increase above background foci was detected. This study does not support compound 1 as a common carcinogen.

Full text of DOI:10.1021/tx3000076

Article
pubs.acs.org/crt
Lactone Metabolite Common to the Carcinogens Dioxane,
Diethylene Glycol, and N-Nitrosomorpholine: Aqueous Chemistry
and Failure to Mediate Liver Carcinogenesis in the F344 Rat
†
†
†
‡
,§
Niangoran Koissi, Niti H. Shah, Brandon Ginevan, William S. Eck, Bill D. Roebuck,*
,
†
and James C. Fishbein*
†Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States
‡Health Effects Research Program, Army Institute of Public Health, U.S. Army Public Health Command, Aberdeen Proving Ground,
Maryland 21010, United States
§
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, United States
ABSTRACT: 1,4-Dioxan-2-one, 1, was synthesized, and the equilibrium constant
between it and the hydrolysis product 2-(2-hydroxyethoxy) acetic acid, 2, was
determined as K = 70 ± 4 in acidic aqueous media, 25 °C, ionic strength 1 M (KCl),
O
and 5% by volume acetonitrile. The carboxylic acid dissociation constant of 2 was
determined under the same conditions to be pK = 3.31 ± 0.02. On the basis of these
a
two determinations, the equilibrium constant between 1 and carboxylic acid anion, 3,
and the proton was calculated to be KOA = 0.034 ± 0.002 M. The stability of 1 was
determined in the range of pH between 1 and 8.5 in buffered aqueous solutions
under the conditions above by UV spectrophotometric methods and exhibited simple
first order kinetics of decay. On the basis of buffer dilution plots, the values of k , the
o
rate constant for solvent mediated decomposition, were determined. The plot of log
k against pH is consistent with a three term rate law for solvolysis with a hydrogen
o
−1
−1
ion catalyzed rate constant k = 1.1 (±0.1) M min , a water catalyzed rate
H+
−
4
−1
4
−1
−1
constant, k = 9.9 (±0.5) × 10 min , and a hydroxide ion catalyzed rate constant, k = 4.1 (±0.3) × 10 M min . The t
1/2
w
OH
for decay at pH 7.0, at 25 °C, is ∼2 h. Treatment of F344 rats with aflatoxin B (AFB ) (positive control) elicited the expected
1
1
preneoplastic foci in the livers of each rat tested, while subsequent administration of 1 (a total of 1.32 g over 12 weeks) failed to
statistically increase focal number or focal volume percent. In 8 rats administered 1 (1.32 g, 12 weeks) alone, no increase above
background foci was detected. This study does not support compound 1 as a common carcinogen.
INTRODUCTION
The powerful rodent liver carcinogen N-nitrosomorpholine,
the much weaker rodent liver carcinogen dioxane,
weak rodent bladder/mammary carcinogen diethyleneglycol
structural analogy with other lactones that manifest DNA
■
11
1
−3
damage, but evidence of genotoxicity by either agent or their
metabolites is weak. In 2007, the U.S. Agency for Toxic
Substances and Disease Registry released the “Draft Toxico-
4
−6
and the
7
,8
14
are all to a significant degree metabolized in vivo to a common
logical Report for 1,4-Dioxane” that summarizes that only 2
of 17 in vitro studies, most employing xenobiotic metabolizing
fractions, elicited positive indications of genotoxicity in the
form of mutation, DNA damage, chromosomal aberrations, or
sister chromatid exchange (SCE). One of these two studies was
considered weakly positive for SCE. A study not included in the
Draft reiterates a lack of mutagenesis in the Ames assay with or
set of compounds (Figure 1) at the carboxylic acid level of
9
−11
oxidation.
Studies with these carcinogens did not identify
all of the metabolites depicted in Figure 1, but their existence
and distribution are dictated by the thermodynamic cycle
depicted within Figure 1. The lactone 1, 1,4-dioxan-2-one
(
Figure 1), was purported to be the major metabolite from
1
1
dioxane in rats. The chemistry and biological activity of 1 is
also of interest as monomeric 1 is used in the synthesis of
15
14
without S9 fraction. As summarized, in vivo studies,
involving doses with rats at, or above, the gram/kilogram
body weight range, are somewhat more positive with respect to
genotoxicity. Five of 15 such studies document mainly
clastogenic activity.
12
polymers employed as resorbable sutures.
The role of lactone 1 in N-nitrosomorpholine carcinogenesis
or its promotion has not been considered. DNA damage from a
major metabolite of N-nitrosomopholine, 3-hydoxy-N-nitro-
somorpholine, has been recently demonstrated via the
formation of covalent adducts with DNA derived from the
The present article in part summarizes work carried out to
determine the stability of lactone 1 preceding its in vivo testing
13
diazonium ion intermediate.
Lactone 1 has been speculated to be a genotoxic agent in
dioxane and dietheylene glycol carcinogenesis, on the basis of
Received: January 4, 2012
Published: March 29, 2012
©
2012 American Chemical Society
1022
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of KCl and hydrogen ion concentration in aqueous solutions that were
5
% by volume acetonitrile and initially contained 0.05 M lactone.
The pKa of (2-hydroxyethoxy)acetic acid was determined by
+
measuring the pH of solutions of the carboxylic acid anion (Na salt)
that were partially neutralized by HCl. The pK measurement was
a
made at various ionic strengths between 0.05 and 1 M with a
concentrated KCl stock. Final solutions contained 5% by volume
acetonitrile. The resulting buffers varied in concentration between 0.07
and 0.08 M. Values of pH were recorded at two temperatures, the
meter standardized at pH 7.00 and 1.10 at the appropriate
temperatures. Average values of pKa were calculated from the
Henderson−Hasselbalch equation. No correction was made for the
existence of the lactone, which would have necessitated changes that
were within the standard deviation of the initial determinations.
Kinetics. The decay of 1 was initially monitored in D O by
2
observing, by 1H NMR, the loss of intensity of the overlapping
resonances (4.78 ppm) for the hydrogens attached to C-6 at 25 °C in
0
.005 M DCl, 0.005 M 1, and 0.005 M acetonitrile as an internal
standard. The kinetics of decay, under identical conditions, of
absorbance at 225 nm was monitored, for comparison, by UV
spectrometry. The good agreement (see Results) of these two
measurements validated an extended kinetic analysis by UV
spectrometry, monitoring absorbance at 220, 225, 230, or 235 nm.
The reaction conditions for the extended study were 5% by volume
Figure 1. Three carcinogens share a common set of metabolites.
and additionally reports its ability to induce or promote liver
carcinogenesis in the rat.
In the F344 rat, hepatic cancer and the process of cancer
formation (i.e., liver carcinogenesis) can be initiated by a
number of chemicals. The liver carcinogen aflatoxin B1 (AFB1)
is a secondary metabolic product of the mold Aspergillus flavus,
acetonitrile in water (H O), ionic strength 1 M (KCl), 25 °C, and 1 at
2
an initial concentration of 0.0013 M. Solutions were buffered with
formic, acetic, cacodylic, and ethylphosphonic acid buffers. Buffer
dilution plots of kobsd against buffer concentration, containing at least 4
buffer concentrations, were constructed from first order decay curves
monitored for at least 3 t1/2 of the reaction. Such plots were linear, and
from extrapolation to zero buffer concentration, the buffer
and AFB is a potent hepatotoxin and carcinogen in the F344
1
rat and many other experimental animals, livestock, and
1
6−19
humans.
The F344 rat is highly sensitive to AFB , and
1
independent rate constant, k , was derived. The associated buffer pH
o
the cancer process (i.e., liver carcinogenesis) has been
extensively studied. The early putative preneoplastic foci that
was taken as the average pH value of the measured values, at reaction
end point, of all the buffer concentrations in a given experiment.
Occasional checks of the pH, at t ∼ 0, indicated no significant change
in pH in the course of the reaction.
20,21
are induced by AFB can readily be quantified,
and both
1
foci size and focal number in the liver can be measured.
Experiments with the F344 rat provide examples of the growth
Synthesis. 1,4-Dioxane-2-one (1). A modification of an
12
22
20,21,23−26
earlier preparation was employed. A 2 L three-neck flask
chilled in an ice bath and containing 134 g of 2-(2-
methoxyethoxy)acetic acid (2 mol) was fitted with a reflux
condenser and a stopper. To the remaining neck of the flask
was fitted a chilled (0 °C) addition funnel containing 113 mL
of AFB -induced foci being enhanced or suppressed
1
by feeding chemicals subsequent to damaging DNA with AFB1.
There is additional evidence that these agents ultimately modify
(
increase or suppress) the occurrence of actual liver cancer and
27,28
not just the precursor foci.
Lactone 1 was evaluated both
(
3 mol equiv) of chilled (0 °C) 62% aqueous hydrobromic acid
for its ability to induce early putative preneoplastic lesions
henceforth termed foci) that ultimately give rise to frank
solution, which was immediately added dropwise to the stirred
round-bottom flask. The reaction mixture was maintained on
ice for another 30 min. The reaction mixture was then stirred at
room temperature for 1 h and subsequently heated with the
temperature gradually rising to reach 150 °C over 2 h. The
temperature was then increased to 180 °C, and the solution was
refluxed overnight. The reaction was subsequently cooled to
room temperature, and the pH was adjusted to 3 with
(
hepatocellular cancers as well as the ability of lactone 1 to
enhance or promote the growth of foci that have been induced
by AFB . In neither case did 1 exhibit measurable activity.
1
EXPERIMENTAL PROCEDURES
■
Chemicals. Except as noted, chemicals, solvents, and isotopically
enriched reagents employed in synthetic and analytical procedures
were obtained from readily available commercial sources. Deionized
water was obtained in house and filtered for HPLC. Acetonitrile was
dried and distilled from calcium hydride. Tetrahydrofuran was dried
and distilled from sodium. HPLC solvents were purchased as HPLC
grade and were not further purified.
NaHCO . The lactone was purified by subsequent vacuum
3
distillation at bp 70−75 °C at 0.8 mmHg to yield a product of
9
tetrahyrofuran yielded 60% (61 g) of colorless needles (T =
0−95% purity based on NMR. Further recrystallization from
m
1
2
4
6
4
6.7 °C). H NMR (400 MHz, CDCl , ppm): δ = 4.49 (t, 2H),
3
.37 (s, 2H), 3.87 (t, 2H). 13C NMR: 166.62, 68.63, 66.42,
2.70. Anal. Calcd for C H O : C, 47.06; H, 5.92. Found: C,
6.90; H, 5.94.
Sodium 2-hydroxyethoxy Acetate (3-Na ). The procedure for the
Analytical Determinations. Measurements of pH were per-
formed on an Orion pH meter, model SA720, with a combination
electrode. Two point calibrations were done before pH values were
recorded. Calibrations were performed using commercially available
standards or 0.10 M HCl, pH 1.10, as appropriate. Elemental analyses
were performed by Atlantic Microlabs, Inc., Norcross, GA.
The equilibrium constant between 1 and its open-chain acid form
was determined by measuring, by means of HPLC, the amount of 1,
calculated by interpolation from a standard curve, that remained after
equilibration of an acidic aqueous solution of 1. The initial
concentration was gravimetrically determined. The equilibrium
constant was determined as a function of ionic strength, by addition
4
6
3
+
synthesis of 1, above, was initiated. Subsequent to the overnight reflux,
the solution was cooled to room temperature and neutralized with
50% aqueous NaOH. The mixture was diluted with 150 mL of
absolute ethanol and heated at 80 °C for 15 min. The solution was
allowed to cool to room temperature with stirring. Stirring was
continued until a white precipitate formed. Crystallization was allowed
to proceed at 4 °C. The white needles were collected and rinsed with
cold absolute ethanol to yield the desired product in 66% final yield.
1
Mp = 206.5 °C. H NMR (400 MHz, D O, ppm): δ = 3.83 (s, 2H),
2
1
023
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3
.60 (t, 2H), 3.50 (t, 2H). 13C NMR: 178.19, 71.80, 60.50. Anal. Calcd
for C H O Na: C, 33.81; H, 4.97. Found: C, 33.84; H, 5.00.
Table 1. Values of K , the Equilibrium Constant for Lactone
1 and Its Open-Chain Acid Form, Determined at 25 °C
o
4
7
4
Animal Studies. Male F344 rats (90−100 g) were purchased from
Charles River Laboratories (Wilmington, MA) and allowed 2 weeks to
acclimatize to the animal facility prior to treatment. Rats were fed the
highly purified diet AIN-76A (American Institute of Nutrition)
without the added antioxidant ethoxyquin (Harlan, Madison, WI).
Throughout the experiments, both food and water were available ad
libitum. The experimental protocol is schematically illustrated in
a
b
[
HCl] (M)
total ionic strength (M)
KO
0
0
0
0
0
0
0
.05
.05
.05
.05
.05
.10
.20
0.05
0.15
0.25
0.45
0.55
1.0
71
72
71
71
70
70
70
1.0
a
Beyond [HCl], the balance of the ionic strength was maintained with
b
KCl. The standard deviation of each determination is <5% on the
basis of triplicate injections.
Values for the pK of 2 under a variety of conditions of ionic
a
strength and temperature are listed in Table 2. The values of
Table 2. Determination of pK Values for 2 under Varying
a
Conditions of Ionic Strength and Temperature
a
b
c
ionic strength (M)
observed pH
calculated pKa
0
0
0
0
.05
.25
.35
.45
3.44 ± 0.01
3.37 ± 0.01
3.37 ± 0.01
3.37 ± 0.01
3.39 ± 0.01
3.43
3.36
3.36
3.36
3.38
Figure 2. Carcinogenesis treatment protocol. Aflatoxin B (AFB )
1
1
treatment is indicated by the arrows (groups 1 and 2), and 1,4-
dioxane-2-one treatment is indicated by the hatched bars (groups 2
and 3). Group 4 is a vehicle treatment group.
0.55
d
e
f
1
.0
.0
3.31, 3.58, 3.81
3.27, 3.54, 3.76
3.31 ± 0.02
3.27 ± 0.01
d
g
h
1
a
Unless otherwise stated, the total buffer concentration was 0.05 M, at
Figure 2. At 5 weeks of age (about 125 g body weight), all rats were
a prepared buffer ratio of AH/A- = 1.0. The balance of the ionic
strength was maintained with KCl. Unless otherwise stated, the value
is the mean and standard deviation of duplicate determinations at 25
gavaged with either AFB (25 μg/rat) or vehicle (tricaprylin) 5 days
1
b
per week for 2 successive weeks for a cumulative dose of 250 μg AFB1
per rat. Commencing the week immediately following AFB treatment,
1
c
°
°
C. Unless otherwise stated, the value is the pK calculated, for T = 25
C, from the observed pH, the buffer ratio, corrected for the fractional
rats were gavaged with lactone 1, dissolved in water immediately prior
to use, three days (MWF) per week for 12 weeks. In the first week, the
doses of dioxane were each 0.02 g/rat for each of the three days. This
a
dissociation of the acid form, and the Henderson−Hasselbalch
equation. For buffer concentrations between 0.070 and 0.080 M.
The balance of ionic strength was maintained with KCl. The pH
values refer to measured values, at 25 °C, for buffers prepared at a
buffer ratio of HA/A- = 1.0, 35/65, and 1/3, respectively. The value of
pK , at 25 °C, is the mean and standard deviation of the pK values
calculated from the observed pH values at the three buffer ratios,
corrected for the fractional dissociation of the acid form, using the
Henderson−Hasselbalch equation. The pH values refer to measured
values, at 37 °C, for buffers prepared at a buffer ratio of HA/A- = 1.0,
35/65, and 1/3, respectively. The value of pK
and standard deviation of the pK
pH values at the three buffer ratios, corrected for the fractional
dissociation of the acid form, using the Henderson−Hasselbalch
equation.
2
9
d
dose was equal to or less than 15% of the reported LD50 dose. In the
subsequent 11 weeks, all doses were 0.04 g/rat, and they were equal to
or less than 26% of the reported LD50 dose.
e
f
At 12 h prior to autopsy, food was withdrawn to reduce the
glycogen accumulation in the livers. From the left lateral hepatic lobe,
multiple 2 mm thick sections were cut by hand, fixed in acetone at 4
a
a
°
C, embedded in paraffin, and processed by routine histological
g
methods. Hepatic sections (5 μm thick) were stained by standard
immunohistochemical methods for expression of glutathione S-
transferase-placental isoform P (GST-P) foci, and the foci were
identified and analyzed by light microscopy. As with previous
h
, at 37 °C, is the mean
values calculated from the observed
a
a
2
1,24,26
analyses,
the observed focal data of the number of foci per
unit area of tissue examined and their individual focal transactional
areas were subjected to morphometric transformation resulting in foci
3
per cm liver tissue, mean focal diameter, and the volume percent of
liver occupied by GST-P positive foci, a parameter analogous to tumor
pK were determined by measuring values of pH of partially
burden. Extensive details of these procedures have been published
a
2
0,26,30
neutralized solutions of the sodium salt of 3, the concentration
of which was gravimetrically determined. On the basis of the
observed pH, the ratio HA/A- was adjusted for fractional
previously.
Statistical Analyses. Focal data (foci per cubic cm, mean
diameter, and volume %) were statistically analyzed using a t test
(
Stata Corp., College Station, TX).
ionization, and the pK was calculated therefrom by use of the
a
Henderson−Hasselbalch equation. No correction was made for
RESULTS
■
the presence of 1 (see Discussion).
Equilibria. Values for the equilibrium constant K (Figure
Kinetics. The kinetics of decay of 1 were initially
O
1
1
) at 25 °C under a variety of ionic strength and acid conditions
are listed in Table 1. The data were obtained by allowing 1 and
to equilibrate, the initial concentration of 1 having been
gravimetrically determined, and measuring the amount of 1
remaining by reverse phase HPLC on the basis of interpolation
from a standard curve for 1.
determined by H NMR by monitoring the disappearance of
hydrogen resonances at position 6 of the lactone in D O
2
2
solutions containing 0.005 M DCl at 25 °C. In addition, decay
of a shoulder of absorbance between 220 and 240 nm was
monitored by UV spectrophotometry under identical con-
ditions. Both processes exhibited good first order decay for 2.5
1
024
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t1/2 of decay (data not shown) and rate constants that were the
Table 3. Observed and Morphometric Data for F344 Rats
Dosed with AFB (Group 1), AFB Followed by 1 (Group
2), 1 Only (Group 3), and Vehicle (Group 4)
−1
same within experimental error: 0.017 ± 0.002 min (NMR)
1
1
−1
and 0.016 ± 0.001 min (UV). The good agreement validated
a more extensive study using the more facile UV method with
observed
morphometric data
reactions in H O solutions containing 5% by volume
2
mean a
diameter
(μm)
acetonitrile.
number
mean foci foci per cubic
Figure 3A depicts the decrease in the UV absorbance
spectrum as a function of time in a cacodylic acid buffer, 90%
a
a
a
group of rats
counted
cm
volume %
1
2
3
4
6
6
8
4
44 ± 8
40 ± 3
0.1 ± 0.1
1230 ± 410
1190 ± 230
6.1 ± 6.1
0
227 ± 35
210 ± 32
65
3.1 ± 1.0
2.1 ± 0.7
0.001
0
0
a
Values reported are mean ± standard error of the mean.
3
(1 only) had a single focus, and that individual focus was
small (65 μm diameter) compared to the average foci induced
by AFB (>200 μm). Rats treated with vehicle only, group 4,
1
had no foci. Observed focal data are inherently biased as large
foci are over represented; thus, these data were subjected to
morphological transformation. For a number of foci per volume
of liver or the mean focal diameter of the foci, there was no
statistically significant difference between groups 1 and 2. Livers
of rats not receiving AFB (groups 3 and 4) had few if any foci.
1
The volume % of liver occupied by GST-P positive foci (i.e.,
focal volume %) is the most robust measure and is analogous to
tumor burden. There was no statistically significant difference
in foci number, size, or volume % between groups 1 and 2 and
groups 3 and 4.
DISCUSSION
■
Equilibria. The equilibrium constant Ko (Table 1) is
comparatively large and in favor of the open chain carboxylic
acid, relative to the lactone. A lower limit value of K > 50, not
o
inconsistent with the values in Table 1, was obtained by
monitoring the disappearance of the lactone multiplets in D O
2
and 1 M DClO to equilibrium. At the end point, the lactone
4
multiplets were barely visible above background preventing the
determination of an actual value. The values determined under
the varying conditions establish that there is a negligible effect
Figure 3. Kinetics of decay of 1,4-dioxan-2-one in aqueous solutions,
5
% by volume acetonitrile, ionic strength 1 M, KCl, cacodylic acid
buffer, and 90% acid form. (A) Change in UV spectrum as a function
of time; total buffer concentration = 0.08 M. (B) Semilogarithmic plot
of absorbance change in A as a function of time, fit to a first order
process is overlaid on the data. (C) Change in kobsd as a function of
buffer concentration extrapolated to the intercept to derive the value of
ko, the buffer independent rate constant.
of varying ionic strength on the equilibrium constant K , as was
o
to have been expected given the absence of charged species on
either side. The values of Ko (Table 1) are slightly to
significantly larger than those reported previously for a set of
31
32
permethylated hexose-δ-lactones and glucono-δ-lactone
which range from 54 to 1.0 in aqueous media. In contrast,
smaller ring lactones can be more thermodynamically favorable
acid form, spanning more than three t1/2 of decay. The change
in absorbance at 225 nm against time for this experiment is
indicated in Figure 3B and includes an overlaid fit to a simple
first-order decay process. Values of kobsd as a function cacodylic
acid concentration are plotted in Figure 3C. Generally these
plots contained a minimum of four points at buffer
concentrations less than 0.2 M total buffer concentration.
Such plots were typically linear and permitted assignment, by
3
3,34
than their open chain forms.
The large value of K means
o
that even in the most favorable, acidic, environment, were the
acid and lactone to reach equilibrium, the concentration of the
lactone would be just 1.4% of the sum of both forms.
The value of the apparent pK for ionization of the open
a
chain acid was determined under differing conditions of ionic
strength and/or temperature on the basis of the measured pH
of partially neutralized solutions of acid. Inspection of the
values in Table 2 suggests expected values and small changes in
extrapolation of a value of k , of the buffer independent rate
o
constant of hydrolysis. For any given plot, the largest value of
kobsd generally did not exceed the value of k by more than a
pK with conditions. In calculating the value, inclusion of the
o
a
factor of 7.
lactone concentration was neglected because the small
Carcinogenesis. The results of the animal studies are
shown in Table 3. Large numbers of GST-P positive foci were
observed in all rats treated with AFB (groups 1 (AFB ) and 2
concentration value affected the absolute value of pK by less
a
than the experimental error in the determinations. By
comparison, the value of the apparent pK of methoxyacetic
1
1
a
(
AFB + 1)), and the average size of these observed foci (focal
acid has been reported as 3.31 at 25 °C, ionic strength 1 M
1
transactional areas) was similar for the two groups treated with
AFB (>200 μm; data not shown). Only one of 8 rats in group
(KCl). Further, the apparent pK values at 25 °C at ionic
a
strength 1 M (NaClO or NaCl) for the permethylated hexose-
1
4
1
025
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Chemical Research in Toxicology
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δ-lactones and glucono-δ-lactone range from 3.31 to
The best fit to the data of the rate law of eq 2 is indicated by
3
1,32,35
4.20.
The effect of changes in ionic strength and
the solid line in Figure 4 with rate constant of k = 1.1 (±0.1)
H+
−1
−1
−4
−1
temperature are small, consistent with expectations on the
M
min , k = 9.9 (±0.5) × 10 min , and k = 4.1
w OH
4
−1
−1
basis of what is known for the effects upon acetic acid
(±0.3) × 10 M min . The best fit to an alternative two-
term rate law containing only terms for the hydrogen and
hydroxide ions is indicated by the dashed line in Figure 4 and
underestimates the rate constants observed in pH range from
3
6,37
ionization.
The determination of K and the pK permits the calculation
o
a
of KOA (Figure.1) as KOA = K × K = 0.034 ± 0.002 M, in
a
o
which the uncertainty is calculated as the error propagated in
the multiplication. Thus, at equilibrium, at pH 7, the lactone
3
.5−6 by rate factors as large ∼4, well outside the experimental
error of the determinations.
−4
represents ∼3 × 10 percent of the total species represented in
The reactivity of lactone 1 with solvent species is comparable
31,32
Figure 1.
with what has been observed for other lactones.
D-Glucono-
Kinetics. The kinetics of decay of lactone 1 were monitored
δ-lactone is more reactive than 1 by less than a factor of 3 in the
hydrogen ion, hydroxide ion, and water-catalyzed hydrolyses.
Similarly, in all but one case, the rate constants for the
hydrolysis reactions of permethylated glycono-δ-lactones are
within a factor of 5 of those determined here for 1. In the case
of tetra-O-methylmannono-δ-lactone, the rate constant for
hydrogen ion catalyzed hydrolysis of 1 is faster by a factor of 12,
though a reason for this is not clear. Surprisingly, 1 is ∼1500
1
in parallel by UV spectrophotometry and HNMR in solutions
of D O, and the values of k
were the same within the error
2
obsd
of the determinations. This validated a more extended
investigation in buffered H O solutions using the more facile
2
UV method.
Generally the value of kobsd varied as a function of buffer
concentration according to eq 1, in which kbuf is the second
order rate constant for acceleration of the decay by total buffer
species, while ko is the buffer-independent rate constant,
obtained by linear extrapolation of plots such as those in Figure
38
more reactive toward hydroxide ion than valerolactone,
perhaps reflecting greater electrophilicity and better leaving
group ability in the former, and possibly a difference in rate
limiting step on the reaction coordinate.
3c.
kobsd = k + kbuf[buffer]total
(1)
Though the equilibria determined above substantially
disfavor lactone 1, the kinetics indicate a chemical stability
that is sufficient to transit the extremes of the digestive tract.
The t1/2 for decay of 1 at pH 2 and 7 is 57 and 127 min,
respectively. The t1/2 at pH 7 is within a factor of 3 of those for
o
While stimulation by the oxygen acid buffers studied was
significant, relative to the buffer independent rate constants, in
the range of buffer concentrations studied, a more detailed
study and analysis of these constants was not attempted.
The variation of the value of log k as a function of solution
pH is presented in Figure 4 in which the solid circles represent
39
the decay of N-nitroso-N-methylurea and N-methyl-N′-nitro-
o
40
N-nitrosoguanidine, while the t1 at pH 2 is within a factor of
3
/2
3
9
of trimethyl-N-nitrosourea. Each of these N-nitroso
compounds is a documented rodent carcinogen when orally
1
,41−43
administered.
These solvolysis studies further established
the feasibility for designing protocols for the animal studies
described below.
Carcinogenesis in the Rat. As expected with this rat
model of liver carcinogenesis, the small total dose and short
dosing schedule of AFB (see Figure 2) resulted in large
1
numbers of hepatic foci (Table 3, group1); however,
subsequent exposure to lactone 1 did not modify the number
of foci (group 1 vs group 2) that developed nor were their
average focal size altered. Additionally, exposure to only lactone
1
resulted in no significant initiation of hepatic foci. One very
small focus was encountered in the tissue sections of 1 of 8 rats
receiving the cumulative total dose of 1.32 g of lactone 1. Small
spontaneous foci are occasionally observed in control rat
30
livers. For example, in a group of control rats exposed to no
known carcinogen, but similar in age and sex to rats of this
study, one spontaneous focus was observed, and it was 2.5
times as large as the one observed in this study.
It is certainly possible that larger doses of lactone 1 treated
over a more lengthy study period with examination of rats
living to much older ages might have provided evidence of
hepatic carcinogenesis, but this study does not presage such an
outcome.
Figure 4. Plot of the log of k as a function of pH. The solid line is a
o
best fit to a three-term rate law, eq 2, for the solvent mediated
hydrolysis. The dashed line is the best fit to a two-term rate law for
solvent mediated hydrolysis.
Alternative genotoxins by which diethylene glycol, dioxane,
and N-nitrosomorpholine might manifest their carcinogenicity
are the aldehyde preceding formation of 1, aldehydes from the
further metabolism of 1−3, and, in the case of N-nitro-
somorpholine, the diazonium ion that has been proven to
alkylate DNA and other aldehydes derived from diazonium ion
the actual values of k determined in this study. The data in
Figure 4 are best described by a rate law for the variation of log
ko with pH that contains three kinetic terms, as indicated in eq
o
2
, one involving the hydrogen ion, k , a pH-independent term,
H+
kw, and one involving hydroxide ion, kOH
.
+
−
ko = kH+[H ] + k + k [OH ]
(2)
44
w
OH
fragmentation.
1
026
dx.doi.org/10.1021/tx3000076 | Chem. Res. Toxicol. 2012, 25, 1022−1028

Chemical Research in Toxicology
Article
(
16) Busby, W. F., and Wogan, G. N. (1984) Aflatoxins, In Chemical
AUTHOR INFORMATION
■
*
Carcinogens (Searle, C. E., Ed.) pp 945−1136, American Chemical
Society, Washington, D.C.
(17) Eaton, W. F., and Groopman, J. D. (1994) The Toxicology of
Aflatoxins: Human Health, Veterinary and Agricultural Significance,
Academic Press, New York.
Corresponding Author
(B.D.R.) Tel: 603-650-1676. Fax: 603-650-1129. E-mail: Bill.
D.Roebuck@dartmouth.edu. (J.C.F.) Tel: 410-455-2190. Fax:
10-455-2608. E-mail: jfishbei@umbc.edu.
4
(18) Kensler, T. W., Groopman, J. D., Sutter, T. R., Curphey, T. J.,
Funding
and Roebuck, B. D. (1999) Development of cancer chemopreventive
This work was supported in part by NIH grant R01 CA52881
to J.C.F.), Aberdeen Proving Ground contract W91ZLK-08-P-
agents: Oltipraz as a paradigm. Chem. Res. Toxicol. 12, 113−126.
(
(19) Kensler, T. W., Roebuck, B. D., Wogan, G. N., and Groopman,
1
038 (to J.C.F.), and NIH grant R01 CA 39416 (to B.D.R.).
J. D. (2011) Aflatoxin: a 50-year odyssey of mechanistic and
translational toxicology. Toxicol. Sci. 120 (Suppl 1), S28−S48.
Notes
(
20) Kensler, T. W., Groopman, J. D., Eaton, D. L., Curphey, T. J.,
The authors declare no competing financial interest.
and Roebuck, B. D. (1992) Potent inhibition of aflatoxin-induced
hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-
dithiole-3-thione. Carcinogenesis 13, 95−100.
ABBREVIATIONS
■
(
21) Roebuck, B. D., Curphey, T. J., Li, Y., Baumgartner, K. J.,
Bodreddigari, S., Yan, J., Gange, S. J., Kensler, T. W., and Sutter, T. R.
2003) Evaluation of the cancer chemopreventive potency of
dithiolethione analogs of oltipraz. Carcinogenesis 24, 1919−1928.
22) Maxuitenko, Y. Y., MacMillan, D. L., Kensler, T. W., and
AFB , aflatoxin B1; HPLC, high-performance liquid chroma-
tography; MWF, Monday−Wednesday−Friday; GST-P, gluta-
1
(
thione S-transferase-placental isoform P
(
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