Microsomal oxidation of N,N-diethylformamide and its effect on P450- dependent monooxygenases in rat liver

Article abstract of DOI:10.1021/tx950201u

N,N-Diethylformamide (DEF) is a hepatotoxic polar solvent in which metabolism has not been investigated. In this study we examined the following: (a) the oxidative metabolism of DEF using both liver microsomes from rats pretreated with selected P450 inducers and purified P450 enzyme (2B1, 2E1, 2C11); and (b) the effect of administration of DEF and its metabolite, the monoethylformamide (MEF), on induction and/or inhibition of the P450 isoforms in rats. DEF was deethylated by microsomal P450-dependent oxidation forming acetaldehyde and MEF according to Michaelis-Menten kinetic parameters. Microsomes from rats pretreated with acetone and pyrazole (selective P4502E1 inducers) or rats pretreated with dexamethasone and 200 mg/kg DEF were able to deethylate DEF in a biphasic manner, showing a low K(m) component with a V(max) of about 0.2 nmol/(min·mg of protein) and a K(m) between 70 μM and 250 μM. The low K(m) component was not present in control microsomes or in microsomes from rats treated with phenobarbital, β- naphthoflavone, or clofibrate, where linear kinetics were observed. The use of purified P4502E1 and 2C11 in a reconstituted system showed that 2E1, which oxidized DEF with a V(max) of 4.5 nmol/(min·nmol of P450) and a K(m) of 0.7 mM, can partially account for the low K(m) DEF deethylase, whereas 2C11, which oxidized DEF with a V(max) of 4.8 nmol/(min·nmol of P450) and a K(m) of 17 mM, might be the high K(m) deethylase. The purified 2B1 was barely able to deethylate DEF. A confirmation of the role of 2E1 in DEF metabolism was obtained by using various selective inhibitors of P450 isoforms and immunoprecipitation experiments with anti P4502E1 IgG. The low K(m) component of DEF deethylation in acetone-or pyrazole-induced microsomes was strongly inhibited (~90%) by diethyldithiocarbamate, 4-methylpyrazole, and anti-2E1 IgG, but in 200 mg/kg DEF-induced microsomes the inhibition was partial, suggesting that other P450(s) may be involved. Administration of DEF 200 mg/kg ip for 4 days induced hepatic microsomal P4502E1-dependent aniline hydroxylase, P4502B1/2-linked pentoxyresorufin O-depentylase, 16β- testosterone hydroxylase P4503A1/2-associated erythromycin N-demethylase, and 6β-testosterone hydroxylase. Alternatively, the same dose regimen of MEF induced only the aniline hydroxylase and depressed the 3A1/2-linked activities. Immunoblot experiments verified these data. These findings indicate that DEF, at low concentrations, is predominantly oxidized by P4502E1 and that this enzyme may be induced in rodents by repeated MEF or DEF treatment, thereby increasing their own metabolism and potentially their cytotoxicity through the formation of ethyl isocyanate.

Full text of DOI:10.1021/tx950201u

8
82
Chem. Res. Toxicol. 1996, 9, 882-890
Micr osom a l Oxid a tion of N,N-Dieth ylfor m a m id e a n d Its
Effect on P 450-Dep en d en t Mon ooxygen a ses in Ra t Liver
Giada Amato, Vincenzo Longo, Arturo Mazzaccaro, and Pier Giovanni Gervasi*
Laboratory of Genetic and Biochemical Toxicology, Istituto di Mutagenesi e Differenziamento,
C.N.R., via Svezia 10, 56124 Pisa, Italy
Received November 29, 1995X
N,N-Diethylformamide (DEF) is a hepatotoxic polar solvent in which metabolism has not
been investigated. In this study we examined the following: (a) the oxidative metabolism of
DEF using both liver microsomes from rats pretreated with selected P450 inducers and purified
P450 enzyme (2B1, 2E1, 2C11); and (b) the effect of administration of DEF and its metabolite,
the monoethylformamide (MEF), on induction and/or inhibition of the P450 isoforms in rats.
DEF was deethylated by microsomal P450-dependent oxidation forming acetaldehyde and MEF
according to Michaelis-Menten kinetic parameters. Microsomes from rats pretreated with
acetone and pyrazole (selective P4502E1 inducers) or rats pretreated with dexamethasone and
2
00 mg/kg DEF were able to deethylate DEF in a biphasic manner, showing a low K
with a Vmax of about 0.2 nmol/(min‚mg of protein) and a K between 70 µM and 250 µM. The
low K component was not present in control microsomes or in microsomes from rats treated
m
component
m
m
with phenobarbital, â-naphthoflavone, or clofibrate, where linear kinetics were observed. The
use of purified P4502E1 and 2C11 in a reconstituted system showed that 2E1, which oxidized
DEF with a Vmax of 4.5 nmol/(min‚nmol of P450) and a K
the low K DEF deethylase, whereas 2C11, which oxidized DEF with a Vmax of 4.8 nmol/(min‚-
nmol of P450) and a K of 17 mM, might be the high K deethylase. The purified 2B1 was
m
of 0.7 mM, can partially account for
m
m
m
barely able to deethylate DEF. A confirmation of the role of 2E1 in DEF metabolism was
obtained by using various selective inhibitors of P450 isoforms and immunoprecipitation
experiments with anti P4502E1 IgG. The low K
or pyrazole-induced microsomes was strongly inhibited (∼90%) by diethyldithiocarbamate,
-methylpyrazole, and anti-2E1 IgG, but in 200 mg/kg DEF-induced microsomes the inhibition
m
component of DEF deethylation in acetone-
4
was partial, suggesting that other P450(s) may be involved. Administration of DEF 200 mg/
kg ip for 4 days induced hepatic microsomal P4502E1-dependent aniline hydroxylase, P4502B1/
2
-linked pentoxyresorufin O-depentylase, 16â-testosterone hydroxylase P4503A1/2-associated
erythromycin N-demethylase, and 6â-testosterone hydroxylase. Alternatively, the same dose
regimen of MEF induced only the aniline hydroxylase and depressed the 3A1/2-linked activities.
Immunoblot experiments verified these data. These findings indicate that DEF, at low
concentrations, is predominantly oxidized by P4502E1 and that this enzyme may be induced
in rodents by repeated MEF or DEF treatment, thereby increasing their own metabolism and
potentially their cytotoxicity through the formation of ethyl isocyanate.
In tr od u ction
N-Alkylformamides, such as diethylformamide (DEF)
and dimethylformamide (DMF), are polar solvents widely
used in the manufacturing of synthetic fibers, leathers,
and films and in chemical laboratories as carriers of
water-insoluble compounds (1, 2).
thought to be responsible for the toxicity of DMF and
NMF (6, 7).
MIC is a potent electrophilic carbamoylating agent
which rapidly (i) conjugates with GSH forming two
thiocarbamates: S-(N-methylcarbamoyl)glutathione (SMG)
and ultimately urinary N-acetyl-S-(N-methylcarbamoyl)-
cysteine (AMCC), both able to perform transcarbamoy-
lating reactions with the nucleophilic cell constituents;
(ii) reacts with water, yielding the urinary methylamine
Hepatotoxicity induced by DMF and by its demethyl
metabolite, N-methylformamide (NMF), has been ob-
served in several species including rats, mice, and
humans (3-5). The mechanism by which DMF and NMF
exert their toxicity was shown to be mediated by their
metabolism (6). DMF is hydroxylated on one methyl
moiety by the P450 system to yield N-(hydroxymethyl)-
1
Abbreviations: AC, acetone; AECC, S-(N-ethylcarbamoyl)-N-ace-
tylcysteine; AMCC, S-(N-methylcarbamoyl)-N-acetylcysteine; AnH,
aniline hydroxylase; APD, aminopyrine demethylase; â-NF, â-naph-
thoflavone; BzD, benzphetamine demethylase; CLO, clofibrate; DEDTC,
diethyldithiocarbamate; DEX, dexamethasone; EIC, ethyl isocyanate;
ErD, erythromycin demethylase; EtD, ethylmorphine demethylase;
EROD, ethoxyresorufin O-deethylase; HEEF, (hydroxyethyl)ethylfor-
mamide; HMF, (hydroxymethyl)formamide; HMMF, (hydroxymethyl)-
methylformamide; MIC, methyl isocyanate; P450, cytochrome P450;
PB, phenobarbital; PROD, pentoxyresorufin O-depentylase; PYR,
pyrazole; PYR-, PB-, DEX-, AC-, âNF-, CLO-, and DEF-microsomes
correspond to hepatic microsomes from PYR-, PB-, DEX-, AC-, âNF-,
CLO-, and DEF-treated rats, respectively; T, testosterone; 17-OT,
1
N-methylformamide (HMMF), which chemically decom-
poses to NMF and formaldehyde. NMF, in turn, may
undergo further P450-mediated oxidation of the N-formyl
group, leading to methyl isocyanate (MIC). This com-
pound and its reactive glutathione (GSH) derivatives are
4
-androstene-3,17 dione; 16R-OH, 16â-OH, 7R-OH, 6R-OH, 6â-OH, 2R-
*
To whom correspondence should be addressed.
Abstract published in Advance ACS Abstracts, J une 15, 1996.
OH, and 2â-OH correspond to 16R-, 16â-, 7R-, 6R-, 6â-, 2R-, and 2â-
hydroxytestosterone, respectively; TAO, triacetyloleandromycin.
X
S0893-228x(95)00201-3 CCC: $12.00 © 1996 American Chemical Society

N,N-Diethylformamide Metabolism
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 883
mark). DEF, MEF, â-NF, DEX, PB, CLO, PYR, DEDTC, and
resorufin were supplied by Fluka (Buchs, Switzerland). En-
zymes and coenzymes were obtained from Boehringer (Man-
nheim, FRG). Ethoxyresorufin and pentoxyresorufin were
synthesized from resorufin by ethylation with ethyl iodide and
by pentylation with pentyl iodide, respectively (15). All other
chemicals and solvents were of analytical grade and were
obtained from commercial sources.
An im a l Tr ea tm en t a n d P r ep a r a tion of Micr osom es.
Male Sprague-Dawley rats (6-8 weeks old, Charles River, FRG)
were injected ip with DEF or MEF at doses of 50, 200, and 400
mg/kg for 4 days. Other inducers administered ip were:
phenobarbital (PB) at 80 mg/kg for 3 days; â-naphthoflavone
(
â-NF) 40 mg/kg for 3 days; dexamethasone (DEX) 50 mg/kg
for 4 days; clofibrate (CLO) 250 mg/kg for 3 days; pyrazole (PYR)
00 mg/kg for 4 days. Acetone (AC) 5% (v/v) was administered
in the drinking water for 10 days.
Animals were killed by CO
asphyxia; the livers were collected
F igu r e 1. Metabolism of DEF.
2
2
and CO ; and (iii) binds nucleophilic sites of cellular
2
macromolecules (7, 8).
and microsomes were prepared as described previously (16). The
washed microsomal pellets were resuspended in 100 mM
phosphate buffer, 1 mM EDTA (pH 7.4), and stored at -80 °C.
Protein content was determined according to Lowry et al. (17)
using bovine serum albumin standards.
Histop a th ologica l Stu d ies. Control male Sprague-Dawley
rats and male CD-1 mice (7-9 weeks old, Charles River, FRG)
were injected ip with DEF (0.5, 1, or 2 g/kg in 0.9% saline).
Animals were killed 48 h after solvent administration; livers
were collected, rapidly fixed in Carnoy fluid, and thereafter
processed for histology.
En zym e Assa ys. Hepatic cytochrome P450 was measured
with the Omura and Sato method (18). Microsomal aniline
hydroxylase (AnH) was determined by measuring the formation
of p-aminophenol as descibed by Ko et al. (19). The aminopyrine
(APD), erythromycin (ErD), benzphetamine (BzD), and ethyl-
morphine (EtD) demethylase activities were assayed by mea-
suring the formation of formaldehyde (20). Ethoxyresorufin
O-deethylase (EROD) and pentoxyresorufin O-depentylation
Gescher’s group (9, 10) has presented evidence that rat
P4502E1, an effective oxidant catalyst of low molecular
weight compounds including many solvents (11), plays a
crucial role in both the hydroxylation of DMF to HMMF
and in the oxidation of HMMF and NMF formyl moiety
to MIC.
There are some indications that the metabolism and
hepatotoxicity of NMF and monoethylformamide (MEF)
are similar (4, 12). In mice, MEF and NMF have been
reported to undergo metabolism to a carbamoylating
intermediate (EIC and MIC) by microsomal oxidation. In
addition, EIC was formed from MEF at a rate faster than
MIC from NMF (13). However, to our knowledge, no data
are provided on the biotransformation of DEF. Regard-
ing its hepatotoxicity, a generic indication of an acido-
phile necrosis caused by DEF treatment in mouse and
rat liver has been reported (14). Although the oc-
cupational exposure to DEF is not as widespread due to
a much lower production of this compound compared to
DMF, studies on DEF can help to better understand the
mechanism of DMF toxicity. Thus, we have studied the
in vitro oxidative metabolism of DEF, postulating that
it is biotransformed by P450 via the same pathway as
DMF (Figure 1).
The first step is the R-hydroxylation of one ethyl moiety
of DEF, which gives rise to N-(hydroxyethyl)ethylforma-
mide (HEEF). This compound is unstable under basic
conditions and decomposes rapidly to acetaldehyde and
MEF. By assaying the formation of acetaldehyde, we
have investigated the role of P450 isoforms in the
oxidation of DEF, using both hepatic microsomes from
rats treated with inducers of selected P450s and three
purified P450s (2B1, 2C11, and 2E1). Since it has been
observed that the substrate for a particular P450 isoform
is also an inducing agent of the same isoenzyme, the
influence of DEF and MEF on P4502E1 and other P450-
dependent monooxygenase activities in rats in vivo was
investigated.
(PROD) activities were determined by measuring the formation
of resorufin in a Perkin-Elmer spectrofluorimeter (21). Test-
osterone hydroxylase was assayed as reported previously (22)
according to an HPLC method described by Platt et al. (23).
DEF deethylation was determined using a method for di-
ethylacetamide (24) by measuring the acetaldehyde formation
after quenching the reaction with NaOH (0.1 M final concentra-
tion). Under alkaline conditions or during GLC analysis, the
HEEF formed as a stable intermediate decomposes to yield
acetaldehyde and MEF. Acetaldehyde was trapped with the
addition of semicarbazide, as described by Yoo et al. (25). The
resulting semicarbazone was reacted with (2,4-dinitrophenyl)-
hydrazine, and its (2,4-dinitrophenyl)hydrazone derivative was
assayed by HPLC as reported previously (16).
Recon stitu ted System . Cytochrome P4502B1 and NADPH-
cytochrome P450 reductase were purified from microsomes of
male Sprague-Dawley rats treated with PB as described previ-
ously (26). P4502E1 was purified from pyrazole-treated diabetic
rats following a procedure similar to that described for hamsters
(27). Rats were made diabetic by a single injection of strepto-
zotocin (90 mg/kg dissolved in 0.1 M citrate buffer, pH 4.5) in
the caudal vein (28). Two weeks after streptozotocin treatment,
diabetes was confirmed by analysis of blood glucose and rats
were treated ip with 150 mg/kg of PYR for 2 days to fortify the
P4502E1 induction (29). P4502C11 was purified from control
male SD rats with the Fujita method (30). The reconstituted
system contained 0.1 nmol of purified P450, 0.3 nmol of P450
reductase, 30 µg of dilauroylphosphatidylcholine (DLPC), and
substrate at the concentration described in 1 mL of 0.1 mM
phosphate buffer, pH 7.4. DLPC was prepared in water and
sonicated immediately before use. After 30 min preincubation
at room temperature, the reaction was started with NADPH (1
mM) and carried out at 37 °C for 30 min. Acetaldehyde was
quantitated by HPLC as previously described (16). The activity
of the purified 2B1 and 2E1 was checked in a reconstituted
system with saturating concentrations of benzphetamine (2B1)
Ma ter ia ls a n d Meth od s
Ch em ica ls. Benzphetamine was from the Upjohn Co.
(Kalamazoo, MI, USA.). Corticosterone, 16â-OH, 17-OT, T,
nitrocellulose filters (0.45 µm), 4-chloro-1-naphthol, and eryth-
romycin were purchased from Sigma Chemicals (St Louis, MO,
USA); 2R-OH, 2â-OH, 6R-OH, 6â-OH, 7R-OH, and 16R-OH were
obtained from the Steroids Reference Collection (D. N. Kirk,
Department of Chemistry, Queen Mary College, London, U.K.).
Rabbit anti-rat P4502B1, P4502E1, and P4503A1 polyclonal
antibodies were obtained from Oxygene (Dallas, TX, USA). Goat
anti-rabbit IgG were purchased from Dako (Copenhagen, Den-

8
84 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Amato et al.
and aniline (2E1) as selective substrates; their turnover num-
bers were 75 and 12 nmol/(min‚nmol of P450), respectively. The
purified 2C11 selectively catalyzed the 2R- and 16R-T hydroxy-
lation with turnover numbers of 3.2 and 3.5, respectively.
Gel Electr op h or esis a n d Im m u n oblottin g. SDS gel elec-
trophoresis was performed using the discontinuous system of
Leammli (31), with a 1.5 mm thick gel and 3% and 7.5%
acrylamide in the stacking and separation gel, respectively.
Proteins were transferred from the slab gel to nitrocellulose
filters following the method of Towbin et al. (32). Immunode-
tections were performed using rabbit anti-rat P4502B1, P4502E1,
and P4503A1 polyclonal antibodies. The bands on the nitrocel-
lulose membranes were quantified by a laser densitometer
(Ultrascan 2202 LKB).
P r ep a r a tion of An tibod ies. Female New Zealand white
rabbits were immunized, as described previously (33), with
P4502E1 purified antigen. Preimmune serum was collected
prior to any injections. For the first immunization, about 0.2
mg of antigen was mixed with complete Freund’s adjuvant. After
6
and 10 weeks, injections of 0.1 mg of antigen in incomplete
Freund’s adjuvant were performed. The immune serum was
collected 10 days after the last injection. Immunoglobulin G
fractions from preimmune serum and immune serum were
purified by precipitation of non-IgG proteins with caprylic acid
at pH 4.5, followed by precipitation of the IgG fraction with
ammonium sulfate at pH 7.4 (34). In immunoblot experiments
the purified anti P4502E1 IgG recognized the same antigen as
the commercial P4502E1 polyclonal antibodies purchased from
Oxygene.
Ch em ica l In h ibition Assa y. Specific P450 inhibitors were
added at 0.1 mM. All the inhibitors were dissolved in methanol,
except sulfaphenazole and metyrapone which were dissolved in
water. As methanol could have an inhibitory effect, it was
F igu r e 2. Effects of DEF administration in liver mice. (A) Liver
of untreated mouse [H & E; original magnification was 85×
(
figure reduced 50% for publication)]. (B) Liver of DEF-treated
evaporated under N
2
. To facilitate the inhibitor redissolution,
mouse (2 g/kg ip) 48 h after administration: an evident
vacuolization involves the centri-mediolobule, while the peri-
lobule is quite normal [H & E; original magnification was 85×
the residue was resuspended in assay buffer by sonication; then
microsomes were added and vortexed vigorously before adding
the other components.
(figure reduced 50% for publication)].
Im m u n op r ecip ita tion Assa y. Microsomes (0.75 mg/mL)
were mixed with different amounts of a given antibody (IgG
fraction) or preimmune rabbit IgG in 100 mM potassium
phosphate buffer (pH 7.4) and allowed to preincubate for 20 min
at 4 °C. Cofactors and substrate were then added and incubated
for 30 min at 37 °C under the conditions previously described.
MEF administration on P450-dependent monooxygenase
activities are shown in Tables 1 and 2.
DEF was administered to rats at doses of 50, 200, and
400 mg/kg/day ip for 4 days (Table 1). At lower dose (50
mg/kg) DEF did not significantly affect either the mi-
crosomal monooxygenase activities AnH, PROD, BzD,
APD, EROD, EtD, and ErD, or the P450 specific content.
E n zym e Kin et ics. Kinetic parameters (K
m
and Vmax) for
DEF deethylation were estimated using a simple computer
program (deviced by Dr. Ambrosetti) designed for a nonlinear
least-squares regression analysis of a Michaelis-Menten equa-
tion. In the case where two enzymes could better explain the
measured rates, the kinetic parameters were estimated by
fitting the data in the following equation (35): V ) Vmax1S/(Km1
At a dose of 200 mg/kg/day, DEF resulted in a
significant increase, compared to control values, of all the
monooxygenase activities tested, except EROD. The
activities of AnH, ErD, and especially PROD linked to
P4502E1, 3A1/2, and 2B1, respectively (37-39) were
enhanced. These changes were also accompanied by a
significant increase in the P450 specific content.
+
S) + Vmax2S/(Km2 + S), where V is the rate of product
formation, S is the substrate concentration, Km1 and Km2 are
the constants for low and high K
m
components, and Vmax1 and
Vmax2 are the maximum rates for the apparent low and high K
m
component, respectively.
At the highest dose (400 mg/kg), DEF did not addition-
ally enhance the monooxygenase activities assayed,
rather some of them (AnH, EtD) and the P450 content
fell back toward the control values, suggesting that this
solvent concentration could be hepatotoxic (Table 1).
Resu lts
Histologica l Effects of DEF Ad m in istr a tion . In
preliminary experiments we have observed that the
administration in rats of a single dose ip of DEF (up to
MEF administration to rats at doses of 50, 200, and
400 mg/kg/day ip for 4 days did not produce a significant
alteration in the P450 specific content (Table 2). At the
lowest dose examined, no increase of the microsomal
monooxygenase activities was observed. At 200 mg/kg
MEF, only the 2E1-dependent AnH activity increased
significantly, whereas the EtD and ErD activities both
associated to P4503A1/2 were significantly decreased. At
the highest dose, MEF resulted in a generalized reduction
of all microsomal monooxygenase activities, possibly as
a consequence of (i) hepatotoxicity, (ii) microsomal P450
destruction, and (iii) inhibition of hemoprotein synthesis.
2
g/kg) did not provoke any clear hepatotoxic effect,
whereas in mice a single dose ip of 1 or 2 g of DEF/kg
was able to result in a modest centrilobular liver damage
(Figure 2). In our experimental conditions, the liver
necrosis was clearly observed in both species only after
MEF (not shown) but not DEF administration. This
pattern of hepatotoxicity of DEF is not surprising as in
both rats and mice similar results for DMF have been
reported (12, 36).
Effects of DEF a n d MEF Ad m in istr a tion on P 450
Activities. Results of studies on the effect of DEF and

N,N-Diethylformamide Metabolism
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 885
Ta ble 1. P 450 Con ten t a n d Mon ooxygen a se Activities in Liver Micr osom es fr om Ra ts Tr ea ted w ith Va r iou s DEF Dosesa
DEF doses (mg/kg)
P450
parameters
enzyme
0
50
200
400
P450
An H
PROD
BzD
APD
EROD
EtD
0.53 ( 0.12
0.55 ( 0.14
0.011 ( 0.005
5.5 ( 1.2
0.69 ( 0.10
0.65 ( 0.07
0.018 ( 0.007
8.6 ( 1.8
0.97 ( 0.11**
1.1 ( 0.15**
0.090 ( 0.016**
14.1 ( 2.5**
11.5 ( 1.4**
0.074 ( 0.02
9.1 ( 1.8*
0.74 ( 0.15
0.51 ( 0.1
0.114 ( 0.02**
15.3 ( 1.3**
9.1 ( 0.7*
0.079 ( 0.02
7.5 ( 0.9*
1.2 ( 0.23*
2E
2B
2B/2C
2B/2C
1A
6.3 ( 1.1
8.2 ( 2.1
0.051 ( 0.015
4.9 ( 0.5
0.75 ( 0.2
0.052 ( 0.008
5.5 ( 2.2
0.86 ( 0.16
3A
3A
ErD
1.4 ( 0.22*
a
Data are presented as means ( of three experiments. Each experiment used microsomes pooled from 3 animals. P450 content is
expressed as nmol/mg of protein and enzymatic activities as nmol/(min‚mg of protein). **Significantly different from control microsomes,
p < 0.01 by Student’s t-test. *p < 0.05.
Ta ble 2. P -450 Con ten t a n d Mon ooxygen a se Activities in Liver Micr osom es fr om Ra ts Tr ea ted w ith Va r iou s MEF Dosesa
MEF doses (mg/kg)
P450
parameters
enzyme
0
50
200
400
P450
AnH
PROD
BzD
APD
EROD
EtD
0.53 ( 0.12
0.55 ( 0.14
0.011 ( 0.005
5.5 ( 1.2
0.71 ( 0.13
0.69 ( 0.18
0.017 ( 0.003
6.5 ( 1.4
0.54 ( 0.11
1.05 ( 0.21*
0.016 ( 0.004
4.4 ( 1.1
0.49 ( 0.08
0.84 ( 0.3
2E
2B
2B/2C
2B/2C
1A
0.007 ( 0.002
3.6 ( 0.7
6.3 ( 1.1
5.9 ( 0.9
4.2 ( 0.8
3.7 ( 0.5*
0.051 ( 0.015
4.9 ( 0.5
0.75 ( 0.2
0.049 ( 0.005
5.1 ( 0.6
0.82 ( 1.5
0.033 ( 0.007
2.9 ( 0.6*
0.17 ( 0.06**
0.031 ( 0.008
2.3 ( 0.5**
0.18 ( 0.07**
3A
3A
ErD
a
Data are presented as means ( SD of three experiments. Each experiment used microsomes pooled from 3 animals. P450 content
is expressed as nmol/mg of protein and enzymatic activities as nmol/(min‚mg of protein). **Significantly different from control microsomes,
p < 0.01 by Student’s t-test. *p < 0.05.
possibly 2B2 (not shown). P4502B2 is known to have
close molecular weight and a high homology (97%) with
2
2
B1 and a cross-reactivity with antibodies raised against
B1 (40). In control microsomes and in microsomes from
MEF-treated rats, the anti-P4502B1 recognized only a
very faint band in keeping with the very low constitutive
level of 2B1 and 2B2 in rat liver (41). Thus, MEF does
not appear to induce hepatic P4502B1/2.
F igu r e 3. Testosterone hydroxylase activities by microsomes
from control (9) and 200 mg/kg DEF- (0) and 200 mg/kg MEF-
As shown in Figure 4, panel B, anti-P4502E1 stained
a protein band in control microsomes in agreement with
the constitutive presence of 2E1 in rat liver (42). In
MEF- and DEF-microsomes, this antibody recognized a
band, corresponding to 2E1, whose intensity increased
with the MEF and DEF doses. Compared to control
microsomes, the 2E1 content, as determined by densito-
metry, increased to 150%, 240%, and 250% after 50, 200,
and 400 mg of MEF/kg treatment and to 140% and 170%
after 50 and 200 mg of DEF/kg treatment. However, in
(
shaded bars) treated rats. Data are means ( SD (bar) of three
experiments. Each experiment used microsomes pooled from 3
animals. Incubations were carried out at 37 °C for 15 min with
1
mg/mL microsomal protein. **Significantly different from
control at p < 0.01 by Student’s t-test; *p < 0.05.
To further investigate the effects of DEF and MEF
administration to rats, the microsomal metabolism of
testosterone, an endogenous substrate selectively oxi-
dized by many P450 isoforms (23), was studied. Neither
DEF nor MEF at the lowest dose (50 mg/kg) did alter
any T hydroxylase activity (data not shown). At 200 mg/
kg, both solvents affected the T hydroxylations (Figure
4
00 mg/kg DEF-microsomes the 2E1 apoprotein content
was depressed to about 80% of the value of control
microsomes in agreement with the lack of induction of
the 2E1-dependent AnH activity observed after the DEF
treatment (see Table 1). Thus, both DEF and MEF seem
to induce the P4502E1 up to a dose of 200 mg/kg.
3
). DEF significantly enhanced the 16â-OH and 17-OT
and 6â-OH T hydroxylations, corresponding to P4502B1/2
16â and 17-OT) and 3A1/2 (6â), respectively (23). On
the contrary, MEF depressed T hydroxylations of the
A1/2-linked 6â-OH, and the 2R-OH, 17-OT and 16R-OH
(
Figure 4, panel C, shows the western blot analysis with
anti-P4503A1. This antibody recognized a wide band
ascribable to 3A1 and 3A2, which are constitutively
present in rat liver and are immunochemically indistin-
guishable as they have a high homology (90%) and the
same molecular mass (43). In microsomes from 200 mg/
kg DEF treated rats, the P4503A1/2 content was en-
hanced by 360%. MEF did not induce the 3A1/2 content
at any dose. Thus, only DEF appears to be an inducer
of P4503A1/2 isoforms at a dose of 200 mg/kg.
3
all associated to the P450 2C11, the major P450 form
constitutively present in rat liver (30).
Im m u n oblot An a lysis. The hepatic microsomal pro-
teins from control and DEF- and MEF-treated rats were
analyzed by western blot using polyclonal antibodies anti-
P450 2B1/2, 2E1, and 3A1/2, in order to check if DEF
and/or MEF treatment could alter the microsomal con-
tent of these P450s.
As illustrated in Figure 4, panel A, in microsomes from
rats treated with DEF at doses of 200 and 400 mg/kg,
anti-P4502B1 recognized a strong and wide band, which
migrates in the same position as purified P4502B1 and
DEF Deeth yla tion by Liver Micr osom es a n d by
P 4502B1, 2E1, a n d 2C11. The results of the metabolic
studies by control or induced microsomal enzymes in the
presence of an NADPH-generating system showed that

8
86 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Amato et al.
F igu r e 4. Western blot analysis of liver microsomes from
control and DEF- and MEF-treated rats. Microsomes (12 µg of
protein in each lane) were subjected to electrophoresis followed
by immunoblot analysis, using polyclonal anti-2B1 IgG (see
panel A), anti-2E1 IgG (see panel B), or anti-3A1 IgG (see panel
C). Lanes 7, 14, and 19 contained microsomes from control rats;
lanes 1, 8, and 15 contained microsomes from 400 mg/kg DEF-
treated rats; lanes 2, 9, and 19 contained microsomes from 400
mg/kg MEF-treated rats; lanes 3, 10, and 17 contained mi-
crosomes from 200 mg/kg DEF-treated rats; lanes 4, 11, and 18
contained microsomes from 200 mg/kg MEF-treated rats; lanes
5
5
, 12 and 6, 13 contained microsomes from 50 mg/kg DEF- and
0 mg/kg MEF-treated rats, respectively.
F igu r e 5. Eadie-Hofstee plot of DEF deethylation catalyzed
by liver microsomes from (A) control and (B) AC-treated rats.
The substrate concentrations ranged from 0.05 to 100 mM; the
individual values were 2, 4, 8, 15, 20, 35, 50, and 100 mM and
DEF was oxidized to an intermediate, most likely being
HEEF, which, under basic conditions, decomposed giving
rise to the DEF deethylation products: acetaldehyde and
MEF (Figure 1). This reaction was catalyzed by P450
as it had an absolute requirement for molecular oxygen
and NADPH; NADH was ineffective as a source of
electrons and in presence of NADPH did not show any
synergy. Carbon monoxide was able, when bubbled into
incubation mixture, to inhibit the acetaldehyde formation
by 86%.
0
.05, 0.1, 0.2, 0.4, 0.6, 2, 5, 12, 20, 50, and 100 mM for control
and AC-microsomes, respectively. V (nmol of CH CHO formed/
3
(
min‚mg of protein)) and S (mM) represent the reaction rate
and substrate concentration, respectively. Experimental details
are described in Materials and Methods.
als and Methods section, we obtained the apparent
kinetic constants illustrated in Table 3. The simple
apparent K
m
for the metabolism of DEF of control
Kin etic P a r a m eter s. In order to establish which
form(s) of P450 were most active in the catalysis on the
deethylation of DEF to acetaldehyde, incubations were
performed using liver microsomes from control, DEF-
treated (200 mg/kg) rats and rats treated with various
classic inducers of the P450 superfamily (PB, AC, PYR,
DEX, âNF, CLO). With control microsomes the dealky-
lation of DEF followed simple Michaelis-Menten kinetics
and was linear up to 30 min and 1.5 mg of microsomal
protein/mL. Also with PB-, âNF-, and CLO-microsomes
microsomes was high (18 ( 6 mM) and not statistically
different from that of PB-microsomes (15 ( 5 mM).
With microsomes from â-NF- and CLO-treated rats,
in which the 1A and 4A P450 subfamilies are strongly
induced (40), kinetic patterns were similar to those of
microsomes from control rats (data not shown), implying
that the above-mentioned P450 enzymes are not involved
in the oxidation of DEF.
The kinetic analysis performed with PYR-, AC-, DEX-,
m
and DEF-microsomes revealed a new low K component
not present in control microsomes with a Km1 ranging
from 70 to 250 µM, i.e., about 2 orders of magnitude lower
(not shown) the DEF deethylation had linear kinetics.
On the contrary, the production of acetaldehyde with AC-,
PYR-, DEX-, and DEF-microsomes, when examined at
substrate concentrations ranging from 0.05 mM (limit of
detection of product) to 100 mM, followed biphasic
kinetics. Typical Eadie-Hofstee plots of DEF deethyla-
tion catalyzed by control and AC-microsomes are il-
lustrated in Figure 5. From the analysis of the DEF
deethylation data performed as described in the Materi-
than the Km2 or the single K
microsomes. The Vmax1 value was ∼0.2 nmol/(mg‚min),
which was about one fourth of the V value seen in
m
observed in uninduced
max
control microsomes. The Vmax2 of DEX-, AC-, PYR-, and
DEF-pretreated microsomes was similar to the Vmax value
of control microsomes. It should be also noted that when
the rates were expressed per nanomole of P450, the

N,N-Diethylformamide Metabolism
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 887
Ta ble 3. Va lu es of Ap p a r en t Kin etic Con sta n ts for DEF Deeth yla tion by P 4502B1, P 4502E1, P 4502C11, a n d Micr osom es
fr om Con tr ol a n d P r etr ea ted Ra tsa
Vmax1
Vmax2
microsomes or
purified P450
major
isoform P450
(nmol/(min‚mg
of protein))
(nmol/(min.mg
of protein))
Km1 (mM)
Km2 (mM)
control
PB
DEX
AC
PYR
18 ( 6
15 ( 5
33 ( 15
28 ( 12
25 ( 8
27 ( 10
0.82 ( 0.23
0.74 ( 0.21
0.97 ( 0.58
1.32 ( 0.36
0.96 ( 0.25
1.22 ( 0.14
2A/2B
3A1
2E1
0.25 ( 0.10
0.07 ( 0.02
0.08 ( 0.03
0.21 ( 0.11
ND
0.21 ( 0.09
0.18 ( 0.05
0.24 ( 0.08
0.19 ( 0.06
0.16
2E1
DEF
P450 2B1
P450 2E1
P450 2C11
0.7
17
4.5
4.8
a
Values are reported as means ( SD for 3 or more experiments performed with different microsomal preparations. Each preparation
contained pooled livers from 2-4 rats. The incubations were performed at 37 °C with a protein concentration of 1 mg/mL. The incubation
of the reconstituted system containing 2B1, 2E1, and 2C11 was carried out as described in Materials and Methods. The activities expressed
as nmol/(min‚nmol of P-450) are the means of two experiments. ND ) not determined. *Significantly different from control microsomes,
p < 0.5 Student’s t-test.
Ta ble 4. Effects of Va r iou s In h ibitor s on Micr osom a l
production of acetaldehyde was significantly lower with
a
Oxid a tion of DEF by AC- a n d DEF -Micr osom es
PB-microsomes than with control microsomes (0.67 (
rate of acetaldehyde
0
.19 versus 1.54 ( 0.45 nmol/(min‚nmol of P450)). This
production (% of control)
inhibitors
0.1 mM)
major
finding may indicate that 2B1 and 2B2, the major PB-
inducible P450 forms along with 2A1, 2C6, and 3A, also
induced by PB (44), are not specifically involved in the
oxidation of DEF.
(
isoform AC-microsomes DEF-microsomes
control
R-NF
metyrapone
DEDTC
sulfaphenazole
-methylpyrazole 2E1
TAO
100
104
83
19
79
100
93
88
51
47
42
92
1A
2B
2E1
2C (?)
Furthermore, these results suggest that P4502E1,
induced in DEF-, AC-, and PYR-microsomes, may account
4
18
85
at least partially for the low K
m
component of DEF
3A
oxidation.
a
The oxidation of DEF was carried out for 30 min at a substrate
concentration of 0.5 mM to study the low Km component of DEF
deethylase in AC- and DEF-microsomes; the specific control
activities were 0.21 and 0.25 nmol/(min‚mg of protein), respec-
tively. The inhibitors were solubilized as described in Materials
and Methods. The data are expressed as means of two experi-
ments.
The apparent involvement of 2E1 in the DEF deethy-
lation is also supported by the finding that this enzyme,
in a reconstituted system, was able to catalyze the
deethylation at a rate about 20-fold higher than that
(Vmax1) observed in AC- or PYR-microsomes (Table 3). The
higher apparent K in the reconstituted system with 2E1
m
compared to Km1 found in PYR- or AC- microsomes
possibly reflects the lack of an important microsomal
component as cytochrome b .
5
Purified 2C11, the most expressed constitutive P450
in rat liver (30), catalyzed the DEF oxidation with a
substantial turnover number but with a high K similar
m
to that of control microsomes. On the contrary, the
purified 2B1 was able to catalyze the reaction at a low
rate (0.16 nmol/(min‚nmol of P450)), suggesting that this
enzyme plays a minor role in DEF oxidation.
In h ibition Stu d ies. Hepatic microsomal oxidation of
DEF was performed at a substrate concentration of 0.5
F igu r e 6. Effect of anti-rat P4502E1 IgG (b) or preimmune
IgG (9) on DEF deethylation by microsomes from AC-treated
rats. The microsomes were preincubated with the IgG fractions
for 20 min. The incubations were performed with 0.5 mM DEF.
Values, which are expressed as a percentage of control activity
m
mM to probe the low K component of DEF deethylase
in both AC- and DEF-microsomes.
(
0.23 nmol/(min‚mg of protein)), are the mean of two experi-
ments.
R-NF-, metyrapone-, and TAO-selective inhibitors for
the P450 1A, 2B, and 3A subfamily, respectively (45-
could be due to the sulfaphenazole-inhibitable P450(s)
presumibly belonging to 2C subfamily.
4
8), did not or only slightly diminished the DEF deethy-
lase activity in either AC- or DEF-microsomes (Table 4).
DEDTC, 4-methylpyrazole, and sulfaphenazole were able
to reduce the DEF deethylase activities, although to a
different extent. It should be noted that both DEDTC
and 4-methylpyrazole are specific rat 2E1 inhibitors (49,
The contribution of 2E1 to the metabolism of DEF was
further evaluated by immunoinhibition experiments. The
0
.5 and 1 mM substrate concentrations were used in
order to saturate only the low K
m
component in AC- and
DEF-microsomes. Increasing amounts of anti-P4502E1
IgG caused a progressive inhibition of acetaldehyde
production from DEF by AC- and DEF-treated rat mi-
crosomes (Figures 6 and 7). However, in the case of AC-
microsomes (Figure 6) the maximum inhibition was about
90%, whereas in DEF-microsomes (Figure 7) the maxi-
mum inhibition was only 70%, supporting the hypothesis
that P450s other than 2E1 may account for the remaining
DEF deethylase activity in DEF-microsomes.
5
0), whereas sulfaphenazole is a potent inhibitor of 2C
enzymes primarily in humans (46); its inhibition efficacy
toward P4502C of rat is not clear. DEDTC and 4-meth-
ylpyrazole were more effective than sulfaphenazole in
AC-microsomes, whereas in DEF-microsomes these agents
inhibited the reaction to 40-50% of control value. These
findings may be indicative of the primary involvement
of 2E1 in the oxidation of DEF in AC-microsomes,
whereas in DEF-microsomes an important contribution

8
88 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Amato et al.
were found decreased by DMF, whereas AnH was in-
creased, suggesting that DMF, like DEF, might be a 2E1
inducer. To our knowledge, no data regarding the effect
of NMF on the P450 system have been reported.
When the in vitro metabolism of DEF was studied, it
was clear that this solvent was oxidized by the microso-
mal P450 system to a stable intermediate, likely HEEF,
which, under basic conditions, decomposed in acetalde-
hyde and MEF, in a time- and protein-dependent man-
ner. A role for P450 in the overall pathway of DEF
oxidation by microsomes was demonstrated by the de-
pendence on oxygen and NADPH and by inhibition by
CO.
Microsomes isolated from AC-, DEF-, DEX-, â-NF-,
PB-, CLO-, and PYR- treated rats were all capable of
deethylating DEF, but only AC-, DEF-, PYR- and DEX-
microsomes did it at rates higher than that of control
microsomes.
In control, PB-, â-NF-, and CLO-microsomes, the
deethylation kinetics of DEF were linear and had a high
Km
(about 15 ( 22 mM). In AC-, DEF-, DEX-, and PYR-
F igu r e 7. Effect of anti-rat P4502E1 IgG (b) or preimmune
IgG (9) on DEF deethylation by microsomes from DEF-treated
rats. The microsomes were preincubated with the IgG fractions
for 20 min. The incubations were performed with 1 mM DEF.
Values, which are expressed as a percentage of control activity
(
0.22 nmol/(min‚mg of protein)), are the mean of two experi-
ments.
Discu ssion
Hepatic P4502E1 plays a key role in the bioactivation
of a variety of environmental low molecular weight
protoxicants and procarcinogens (11), including benzene
(
51), N-nitrosodimethylamine (25), N-nitrosodiethy-
lamine (52), pyridine (53), DMF, and NMF (10).
The objectives of this study were to elucidate the
oxidative biotransformation of DEF (an alkyl homologue
of DMF) and to verify whether DEF was metabolized
predominantly by P4502E1 and if DEF and/or its proxi-
mal metabolite, MEF, were able to induce P4502E1.
These solvents, when administered acutely to rats, mark-
edly affected some hepatic microsomal monooxygenase
activities. DEF and MEF treatment depressed the P450
activities at doses of 400 mg/kg, probably due to hepa-
totoxicity.
microsomes, this deethylation exhibited two separate
kinetic curves, indicating the existence of at least two
P450 isoenzymes that dealkylate DEF with different
affinities. The P450 responsible for the appearance in
AC- and PYR-microsomes of a new low Km component
for DEF deethylase (Km 70-80 µM) seems to be the
P4502E1, which was strongly induced in these mi-
crosomes. In DEX-microsomes the P450(s) responsible
remain(s) veiled, as the 2B1/2 and 3A induction (47) does
not appear to have an important role. In DEF-mi-
crosomes, in which 2E1 was moderately induced, the
contribution of this enzyme can account at least partially
for the low Km component (Km ) 0.21 mM) of DEF
deethylase.
At concentrations of 200 mg/kg, DEF increased the
amount of P450 content and induced the P4502B1/2,
3
A1/2 and 2E1. The induction of 2B1/2 was demon-
strated by a significant increase in the 2B1/2-dependent
PROD and 16â-OH T hydroxylation activities. The
induction of 2E1 and 3A1/2 was demonstrated by a
significant enhancement of the 2E1-linked AnH and the
The constitutive modest presence of 2E1 (5-8%) in the
microsomal P450 population of control adult rat liver (42)
and the HPLC limits of acetaldehyde detection do not
allow detection of a very low DEF deethylase activity in
control microsomes (<50 pmol/(min‚mg of protein)). A
similar low Km component (Km ) 0.2 mM), at least
partially dependent on 2E1, was also reported for the
microsomal biotransformation of DMF to HMMF (9).
Furthermore, it was observed, that at saturating sub-
strate concentrations (DMF ) 10 mM) for this low Km
component, the DMF demethylation rate in hepatic
microsomes from AC-treated rats was about 3 times that
of control microsomes (9). Thus, the activity of 2E1
toward DMF oxidation could be higher than that found
for the oxidation of DEF. Of course, aside from 2E1, the
contribution of other unidentified isozymes can also occur
in the oxidative metabolism of DMF.
3
A1/2-mediated EtD, ErD, 2â-OH, and 6â-OH T hy-
droxylation activities, respectively. At a concentration
of 50 mg/kg, DEF did not induce the activity of any
monooxygenase including the 3A-linked ErD and the
most inducible 2B1-dependent PROD and 16â-T hy-
droxylase. Thus DEF, as compared to PB, is a modest
inducer of 2B and 3A isoforms. Indeed, the ip adminis-
tration in rats for 4 days of an equimolar dose of PB
produces a various-fold induction of the above-mentioned
activities (data not shown).
MEF, unlike DEF, at doses of 200 mg/kg induced only
the AnH and selectively depressed the EtD and ErD
activities. The reason for the selective depression of these
3
A-dependent activities is not known, but it could be
That 2E1 is a principal catalyst of DEF deethylation
at low substrate concentration is further supported by
(a) the selective inhibition by DEDTC and 4-methylpyra-
zole, quite specific inhibitors for 2E1, (b) the high
catalytic turnover exhibited, in a reconstitutive system,
by purified rat liver 2E1, and (c) the complete inhibition
(90%) of DEF deethylase activity by anti-P4502E1 in AC-
microsomes and partial inhibition (70%) in DEF-mi-
crosomes. On the other hand, 2B1, also inducible by
DEF, appeared to exert a minor role on DEF deethylase,
as purified 2B1 showed a low turnover and metyrapone
linked to the P4503A subfamily functioning as suicide
enzyme in the biotransformation of MEF.
Immunodetectable P4502B1/2, 3A1/2, and 2E1 were
elevated in microsomes from 200 mg/kg DEF-treated
rats, consistent with the alteration of the above mo-
nooxygenase activities. In the MEF-microsomes only the
amount of 2E1 apoprotein was elevated. Thus, DEF, like
AC and other solvents (41), appears to be an inducer of
both 2E1 and 2B1, whereas MEF appears to be a specific
2
E1 inducer. Unlike DEF, it has been demonstrated that
acute administrations of DMF to rats did not increase
or rather decreased, depending on the dose (36, 54), the
microsomal concentration of P450. The 2C-mediated
aminopyrine demethylase along with other activities
elicited a scanty inhibition. Regarding the high K
m
component of DEF deethylation, the 2C11 appeared to
play an important role. The purified 2C11 was found to
catalyze the DEF oxidation with a high turnover but with

N,N-Diethylformamide Metabolism
a high K (17 mM) similar to that of control microsomes.
Thus, this enzyme may account at least partially for the
DEF deethylase activity in untreated microsomes and for
the high K component in microsomes from induced rats.
m
Of course, other P450 constitutive isoforms and especially
the major ones belonging to 2C subfamily (2C6, 2C7,
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 889
(10) Hyland, R., Gescher, A., Thummel, K., Schiller, C., J heeta, P.,
Mynett, K., Smith, A. W., and Mraz, J . (1992) Metabolic oxidation
and toxification of N-methylformamide catalyzed by the cyto-
chrome P450 isoenzyme CYP2E1. Mol. Pharmacol. 41, 259-266.
(11) Guengerich, F. P., Kim, D. H., and Iwasaki, M. (1991) Role of
human cytochrome P4502E1 in the oxidation of many low
molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168-
m
1
79.
2
C13) may contribute to this reaction.
(
12) Kestell, P., Threadgill, M. D., Gescher, A., Gledhill, A. P., Shaw,
A. J . and Farmer, P. B. (1987) An investigation of the relationship
between hepatotoxicity and the metabolism of N-alkylformamides.
J . Pharmacol. Exp. Ther. 240, 265-270.
13) Cross, H., Dayal, R., Hyland, R., and Gescher, A. (1990) N-
Alkylformamides are metabolized to N-alkylcarbamoylating spe-
cies by hepatic microsomes from rodents and humans. Chem. Res.
Toxicol. 3, 357-362.
14) Chann, P. H., Xuong, N. D., and Arnn-Gelade, M. C. (1971) Etude
toxicologique de la formamide et de ser d e´ riv e´ s N. M e´ thyl e´ s et
N. Ethyl e´ s. Th e´ rapie 26, 409-429.
15) Klotz, A. V., Stogeman, J . J ., and Walsh, C. (1984) An alternative
7-ethoxyresorufin assay: a continuous visible spectrophotometric
method for measurement of cytochrome P450 monooxygenase
activity. Anal. Biochem. 140, 138-145.
It is well documented that many compounds augment
the rate of their own metabolism by elevating the activity
of certain P450 enzymes. The data presented here
demonstrate that 2E1 has a major contribution to the
high affinity component of DEF metabolism and that 2E1
is inducible by DEF and its proximal metabolite, MEF.
Thus, reiterative or continuous exposure to DEF could
potentiate its own oxidative metabolism and potentially
its bioactivation to toxic intermediates mainly through
the induction of P4502E1. It remains to be elucidated if
HEEF and MEF are both oxidized to EIC by 2E1 as in
the case of HMMF and NMF (9).
In this regard, it is worth noting that MEF, in mice,
has been found to undergo more rapid biotransformation
than NMF, yielding higher amounts of the corresponding
alkylamine, mercapturic acid, and unchanged parent
compound (12). Possibly, this reflects a faster formation
of EIC from MEF than MIC from NMF.
(
(
(
(
16) Longo, V., Citti, L., and Gervasi, P. G. (1986) Metabolism of
diethylnitrosamine by nasal mucosa and hepatic microsomes from
hamster and rat: species specificity of nasal mucosa. Carcino-
genesis 7, 1323-1328.
(
17) Lowry, O. H., Rosebrough, N. J ., Farr, A. L., and Randall, R. J .
(
1951) Protein measurement with the Folin phenol reagent. J .
Biol. Chem. 193, 265-275.
(18) Omura, T., and Sato, R. (1964) The carbon-monoxide binding
protein of liver microsomes. I. Evidences for its hemoprotein
nature. J . Biol. Chem. 293, 2370-2378.
19) Ko, I. Y., Park, S. S., Song, B. J ., Patten, C., Tan, Y., Han, Y. C.,
Yang, C. S., and Gelboin, H. V. (1987) Monoclonal antibodies to
ethanol-induced rat liver cytochrome P450 that metabolize aniline
and nitrosamines. Cancer. Res. 47, 3101-3109.
20) Tu, Y. Y., and Yang, C. S. (1983) High-affinity nitrosamine
dealkylase system in rat liver microsomes and its induction by
fasting. Cancer. Res. 43, 623-629.
Thus, the link between the P450 isoforms involved in
the oxidation of the above solvents, and the production
of corresponding alkyl isocyanates, warrants further
study, especially in humans. It could be the case that
humans are particularly prone to DEF and MEF hepa-
totoxicity.
(
(
Ack n ow led gm en t. We acknowledge Dr. M. Minks
for critical review of the manuscript, Dr. E. Chieli for the
histological data, and Dr. R. Ambrosetti for having
devised the computer program for the analysis of enzyme
kinetics. This work was partially supported by “Progetto
Finalizzato FATMA” of C.N.R.
(21) Krijgsheld, K. R., and Gram, T. E. (1984) Selective induction of
renal microsomal cytochrome P450-linked monooxygenases by
1
1
,1-dichloroethylene in mice. Biochem. Pharmacol. 33, 1951-
956.
(
22) Longo, V., Mazzaccaro, A., Naldi, F., and Gervasi, P. G. (1991)
Drug metabolizing enzymes in liver olfactory and respiratory
epithelium of cattle. J . Biochem. Toxicol. 6, 123-128.
(23) Platt, K. L., Molitor, E., Dohemer, J ., Dogra, S., and Oesch, F.
(
1989) Genetically engineered V79 Chinese hamster cell expres-
Refer en ces
sion of purified cytochrome P450IIB1 monooxygenase activity. J .
Biochem. Toxicol. 4, 1-6.
(
(
1) IARC (1989) Dimethylformamide. IARC Monogr. 47, 171-197.
2) Katritzky, A. R., Chang, H. X., and Yang, B. (1995) N-Formyl-
benzotriazole: a stable and convenient N- and O-formylating
agent. Synthesis 5, 503-505.
(
24) Menicagli, S., Longo, V., Mazzaccaro, A., and Gervasi, P. G. (1994)
Microsomal metabolism of N,N-diethylacetamide and N,N-dim-
ethylacetamide and their effects on drug-metabolizing enzymes
of rat liver. Biochem. Pharmacol. 48, 717-726.
25) Yoo, J . N., Ishizaki, H. and Yang, C. S. (1990) Roles of cytochrome
P450IIE1 in the dealkylation and denitrosation of N-nitrosodim-
ethylamine and N-nitrosodiethylamine in rat liver. Carcinogenesis
(
(
(
3) Mraz, J ., Cross, H., Gescher, A., Threadgill, M. D., and Flek, J .
(
(
1989) Differences between rodents and human in the metabolic
toxification of N,N-dimethylformamide. Toxicol. Appl. Pharmacol.
8, 507-516.
9
1
1, 2239-2243.
4) Shaw A. J ., Gescher, A., and Mraz, J . (1988) Cytotoxicity and
metabolism of the hepatotoxin N-methylformamide and related
formamides in mouse hepatocytes. Toxicol. Appl. Pharmacol. 95,
(
26) Guengerich, F. P. (1982) Microsomal enzymes involved in toxicol-
ogy-analysis and separation. In Principles and Methods of Toxi-
cology (Hayes, W., Ed.), pp 609-634, Raven Press, New York.
27) Puccini, P., Menicagli, S., Longo, V., Santucci, A., and Gervasi,
P. G. (1992) Purification and characterization of an acetone-
inducible cytochrome P450 from hamster liver microsomes. Bio-
chem. J . 287, 863-870.
28) Donahue, B. S., and Morgan, E. T. (1990). Effects of vanadate on
hepatic cytochrome P450 expression in streptozotocin-diabetic
rats. Drug Metab. Dispos. 18, 519-526.
1
62-170.
(
5) Kennedy, G. L. (1985) Biological effect of acetamide, formamide,
and the monomethyl and dimethyl derivatives. CRC Crit. Rev.
Toxicol. 17, 129-182.
6) Gescher, A. (1993) Metabolism of N,N-dimethylformamide: key
to understanding of its toxicity. Chem. Res. Toxicol. 6, 245-251.
7) Slatter, J . G., Rashed, M. S., Pearson, P. G., Han, D.-H., and
Baillie, T. A. (1991) Biotransformation of methyl isocyanate in
the rat. Evidence for glutathione conjugation as a major pathway
of metabolism and implications for isocyanate-mediated toxicities.
Chem. Res. Toxicol. 4, 157-161.
8) Threadgill, M. D., Axworthy, D. B., Baillie, T. A., Farmer, P. B.,
Farrow, K. C., Gescher, A., Kestell, P., Pearson, P., and Shaw, A.
J . (1987) Metabolism of N-methylformamide in mice: primary
kinetic deuterium isotope effect and identification of S-(N-
methylcarbamoyl)glutathione as a metabolite. J . Pharmacol. Exp.
Ther. 242, 312-319.
9) Mraz, J ., J heeta, P., Gescher, A., Hyland, R., Thummel, K., and
Threadgill, M. D. (1993) Investigation of the mechanistic basis
of N,N-dimethylformamide toxicity. Metabolism of N,N-dimeth-
ylformamide and its deuterate isotopomers by cytochrome P4502E1.
Chem. Res. Toxicol. 6, 197-207.
(
(
(
(
29) Wu, D., and Cederbaum, A. I. (1993) Combined effects of strep-
tozotocin-induced diabetes plus 4-methylpyrazole treatment on
rat liver cytochrome P4502E1. Arch. Biochem. Biophys. 302, 175-
1
82.
(
(30) Fujita, S., Morimoto, R., Chiba, M., Kitani, K., and Suzuki, T.
(1989) Evaluation of the involvement of a male specific cytochrome
P450 isozyme in a senescence-associated decline of hepatic drug
metabolism in male rats. Biochem. Pharmacol. 38, 3225-3231.
(31) Laemmli, U. K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680-685.
(32) Towbin, H., Stachelin, P., and Gordon, J . (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl. Acad. Sci.
U.S.A. 76, 4350-4354.
(

8
90 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Amato et al.
(
33) Kaminsky, L. S., Fasco, M. J ., and Guengerich, F. P. (1981)
Production and application of antibodies to rat liver cytochrome
P-450. Methods Enzymol. 74, 262-272.
(45) Murray, M., and Reidy, G. F. (1990) Selectivity in the inhibition
of mammalian cytochromes P450 by chemical agents. Pharmacol.
Rev. 42, 85-101.
(
34) McKinney, M. M., and Parkinson, A. (1987) A simple, non
chromatographic procedure to purify immunoglobins from serum
and ascites fluid. J . Immunol. Methods 96, 271-278.
35) Dixon, M., and Webb, E. C. (1972) In Enzimi, pp 85, Istituto
Editoriale Universitario, Milano.
(
(
(
46) Halpert, J . R., Guengerich, F. P., Bend, J . R., and Correia, M. A.
(1994) Contemporary issue in toxicology. Selective inhibitors of
cytochrome P450. Toxicol. Appl. Pharmacol. 125, 163-175.
(
(
(
47) Namkung, M. J ., Yang, H. L., Hulla, J . E., and J uchau, M. R.
(1988) On the substrate specificity of cytochrome P4503A1. Mol.
36) Scailteur, V., and Lauwerys, R. R. (1987) Dimethylformamide
Pharmacol. 34, 628-637.
(DMF) hepatotoxicity. Toxicology 43, 231-238.
48) Roos, P. H., Golub-Ciosk, B., Kallweit, P., Kauczinski, D., and
Hanstein, W. B. (1993) Formation of ligand and metabolite
complexes as a means for selective quantitation of cytochrome
P450 isozymes. Biochem. Pharmacol. 45, 2239-2250.
37) Reinke, L. A., Sexter, S. H., and Rikans, L. E. (1985) Comparison
of ethanol and imidazole pretreatments on hepatic monooxyge-
nase activities in the rat. Res. Commun. Chem. Pathol. Pharma-
col. 47, 97-106.
(
38) Lubet, R. A., Mayer, R. T., Cameron, J . W., Nims, R. W., Burke,
M. D., Wolf, T., and Guengerich, F. P. (1985) Dealkylation of
pentoxyresorufin: a rapid and sensitive assay for measuring
induction of cytochrome(s) P450 by phenobarbital and other
xenobiotic in rat. Arch. Biochem. Biophys. 238, 43-48.
39) Arlotto, M. P., Sonderfan, A. J ., Klassen, C. D., and Parkinson,
A. (1987) Studies on the pregnenolone-16R-carbonitrile-inducible
of rat liver microsomal cytochrome P450 and UDP-glucuronyl-
transferase. Biochem. Pharmacol. 36, 3859-3866.
(49) Brady, J . F., Xiao, F., Wang, M. H., Li, Y., Ning, S. H., Gapac, J .
M., and Yang, C. S. (1991) Effects of disulfiram on hepatic
P4502E1, other microsomal enzymes and hepatotoxicity in rats.
Toxicol. Appl. Pharmacol. 108, 366-373.
(
50) Feierman, D. E., and Cederbaum, A. I. (1987) Increased sensitivity
of the microsomal oxidation of ethanol to inhibition by pyrazole
and 4-methylpyrazole after chronic ethanol treatment. Biochem.
Pharmacol. 36, 3277-3283.
(
(
51) Nakajima, T., Wang, R. S., Elovaara, E., Park, S. S., Gelboin, H.
V., and Vainio, H. (1992) A comparative study on the contribution
of cytochrome P450 isozymes to metabolism of benzene, toluene,
and trichloroethylene in rat liver. Biochem. Pharmacol. 43, 251-
(
(
(
40) Ryan, D. E., and Levin, W. (1990) Purification and characteriza-
tion of hepatic microsomal cytochrome P450 Pharmacol. Ther. 45,
1
53-239.
41) Imaoka, S., Terano, Y., and Funae, Y. (1990) Changes in the
amount of cytochrome P450s in rat hepatic microsomes with
starvation. Arch. Biochem. Biophys. 278, 168-178.
42) J ohansson, I., Ekstrom, G., Sholte, B., Puzycki, D., J ornvall, H.,
and Ingelman-Sundberg, M. (1988) Ethanol-fasting and acetone-
inducible cytochrome P450 in rat liver: replication and charac-
terization of enzymes belonging to the IIB and IIE gene subfami-
lies. Biochemistry 27, 1925-1934.
2
57.
(
52) Puccini, P., Fiorio, R., Longo, V., and Gervasi, P. G. (1989) High
affinity diethylnitrosamine-deethylase in liver microsomes from
acetone-induced rats. Carcinogenesis. 10, 1629-1634.
(53) Kim, S. G., Philpot, R. M., and Novak, R. F. (1991) Pyridine effects
on P4502E1, 2B and IVB expression in rabbit liver: characteriza-
tion of high- and low-affinity pyridine N-oxygenases. J . Pharma-
col. Exp. Ther. 259, 470-477.
(
43) Gemzik, B., Greenway, D., Nevins, C., and Parkinson, A. (1992)
Regulation of two electrophoretically distinct proteins recognized
by antibody against rat liver cytochrome P4503A1. J . Biochem.
Toxicol. 7, 43-52.
(
54) Imasu, K., Fujishiro, K., and Inoue, N. (1992) Effects of dimeth-
ylformamide on hepatic microsomal monooxygenase system and
glutathione metabolism in rats. Toxicology 72, 41-50.
(
44) Waxman, D. J ., and Azaroff, L. (1992)Phenobarbital induction of
P450 gene expression. Biochem. J . 281, 577-592.
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