Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes

Article abstract of DOI:10.1021/tx000043t

For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system, the benzamidoxime reductase, has been described. To reconstitute the complete activity of the benzamidoxime reductase, the system required cytochrome b₅, NADH-cytochrome b₅-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic process. It was of interest to study the possibility of benzamidoxime reductase reducing N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall, N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines ofmethamphetamine, amphetamine, and N-methylamine as model compounds were investigated. Formation of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime reductase to their parent amines with reduction rates of 220.6 nmol min^-1 (mg of protein)^-1 for methamphetamine, 5.25 nmol min^-1 (mg of protein)^-1 for amphetamine, and 153 nmol min^-1 (mg of protein)^-1 for N-methylhydroxylamine. Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydroxylamines.

Full text of DOI:10.1021/tx000043t

Chem. Res. Toxicol. 2000, 13, 1037-1045
1037
Red u ction of Am p h eta m in e Hyd r oxyla m in e a n d Oth er
Alip h a tic Hyd r oxyla m in es by Ben za m id oxim e Red u cta se
a n d Hu m a n Liver Micr osom es
Bernd Clement,*,† Detlef Behrens,† Wenke Mo¨ller,† and J ohn R. Cashman‡
Pharmazeutisches Institut, Christian-Albrechts-Universita¨t zu Kiel, Gutenbergstrasse 76,
D-24118 Kiel, Germany, and Human BioMolecular Research Institute, 5310 Eastgate Mall,
San Diego, California 92121
Received February 24, 2000
For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as
amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system,
the benzamidoxime reductase, has been described. To reconstitute the complete activity of the
benzamidoxime reductase, the system required cytochrome b5, NADH-cytochrome b5-reductase,
and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to
homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing
aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic
process. It was of interest to study the possibility of benzamidoxime reductase reducing
N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall,
N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of
medicinally relevant compounds, the hydroxylamines of methamphetamine, amphetamine, and
N-methylamine as model compounds were investigated. Formation of methamphetamine and
amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines
were easily reduced by benzamidoxime reductase to their parent amines with reduction rates
of 220.6 nmol min-1 (mg of protein)-1 for methamphetamine, 5.25 nmol min-1 (mg of protein)-1
for amphetamine, and 153 nmol min-1 (mg of protein)-1 for N-methylhydroxylamine.
Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary
rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime
of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells.
The benzamidoxime reductase is therefore involved in the detoxication of these reactive
hydroxylamines.
could not be detected (5). In previous studies, a pig liver
microsomal hydroxylamine reductase was described by
Kadlubar and Ziegler (6), and that system had many
characteristics of the benzamidoxime reductase described
herein.
The N-oxygenation of alkylamines is a well-known
metabolic process (7). If N-oxygenated metabolites were
reduced back to the parent amines, this would constitute
another example of a futile metabolic cycle involving
oxidation and reduction (Scheme 1).
Methamphetamine (Scheme 1) and amphetamine
(Scheme 1) derivatives are used therapeutically for
attention hyperactivity deficit disorder in children and
as short-term adjunct in exogenous obesity. Amphet-
amine is also used in the treatment of narcolepsy and in
the short-term managment of depression in patients
intolerant to the tricyclic antidepressants (8).
Metabolism of methamphetamine in humans and
animals produces a number of urinary metabolites,
including amphetamine, norephedrine, 4-hydroxynorephe-
drine, N-hydroxymethamphetamine and phenylacetone,
benzoic acid, and hippuric acid (9-12).
In tr od u ction
In previous studies, it was shown that N-hydroxylated
derivatives of strongly basic functional groups were
reduced both in vivo and in vitro by a microsomal enzyme
system present in all mammalian species (rats, rabbits,
pigs, and humans) tested to date (1-4). An enzyme
system that required NADH-cytochrome b5-reductase,
cytochrome b5, and a third protein component, named the
benzamidoxime reductase, was shown to be responsible
for the microsomal enzymatic reductions (5). The third
enzyme component of the reductase system exhibited all
the characteristics of a cytochrome P450 enzyme (5).
Benzamidoxime reductase, isolated from pig liver mi-
crosomes, exhibited similarities with other isoenzymes
of the cytochrome P450 2D subfamily from other species.
Cytochrome P450 2D enzymes from pig have not been
described previously (5). Benzamidoxime reduction activ-
ity in the presence of microsomes from cell lines trans-
fected with cDNAs expressing human cytochrome P450
enzymes obtained from GENTEST Corp. (Woburn, MA)
* To whom correspondence should be addressed: Pharmazeutisches
Institut, Christian-Albrechts-Universita¨t zu Kiel, Gutenbergstrasse 76,
D-24118 Kiel, Germany. Telephone: +431/880-1126. Fax: +431/880-
1352. E-mail: bclement@pharmazie.uni-kiel.de.
Microsomal preparations from human livers catalyze
the NADPH-dependent N-oxygenation of methamphet-
amine to N-hydroxymethamphetamine. It was reported
that in animals the N-hydroxylation of methamphet-
† Christian-Albrechts-Universita¨t zu Kiel.
‡ Human BioMolecular Research Institute.
10.1021/tx000043t CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/23/2000

1038 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Clement et al.
P r ep a r a tion of Meth a m p h eta m in e Hyd r oxyla m in e a n d
Am p h eta m in e Hyd r oxyla m in e. The synthesis of amphet-
amine hydroxylamine and methamphetamine hydroxylamine
was as previously described (14).
Sch em e 1. Meta bolic Cycle of th e Meta bolism of
Alip h a tic Am in esa
P ig Liver Micr osom es. Pig liver microsomes were prepared
by fractional acid precipitation according to the procedure of
Ziegler and Pettit (20) with slight modifications (21).
Hu m a n Liver Micr osom es. Human liver was obtained from
the Medical Department of the University of Kiel. Prior consent
of the local medical ethics committee and from the donors before
removal of the liver pieces was granted. Liver pieces from partial
hepatectomies (Chirurgische Klinik der CAU, Kiel, Germany)
were minced with knives and washed with 20 mM potassium
phosphate buffer containing 0.25 mM sucrose (pH 7.4) at 4 °C.
The minced livers were homogenized using a motorized Teflon
pestle glass tube homogenizer (Potter S, for 30 mL, Braun
Melsungen AG, Melsungen, Germany). After adjustment to pH
7.4, the homogenates were transferred to plastic tubes and
centrifuged at 9000g for 30 min. The supernatant was carefully
decanted and centrifuged at 100000g for 60 min. The pellet of
the 100000g centrifugation was resuspended in phosphate buffer
(pH 7.4) and again centrifuged at 100000g for 60 min. The
supernatant was discarded, and the microsome pellet was
resuspended in potasium phosphate buffer (pH 7.4).
a
(A) Reduction by benzamidoxime reductase, composed of
cytochrome b5, NADH-cytochrome b5-reductase, and
enzyme, the benzamidoxime reductase. (B) Hydroxylation by P450
and the flavin-containing monooxygenases (12-14, 18).
a P450
The microsomal preparation was stored at -80 °C in aliquots.
P u r ifica tion of Cytoch r om e b5, NADH-Cytoch r om e b5-
Red u cta se, a n d Ben za m id oxim e Red u cta se. Cytochrome b5,
NADH-cytochrome b5-reductase, and benzamidoxime reductase
were fractionated by the procedure described by Clement et al.
(5) with slight modifications. Thesit (Boehringer Mannheim,
Mannheim, Germany) was used to solubilize the microsomal
proteins in the elution buffers. All purification steps were
performed at 4 °C. The solubilized pig liver microsomes were
applied to an octyl-sepharose CL 4B (Pharmacia, Freiburg,
Germany) column (38 cm × 3.8 cm), and enzymes were eluted
as described in detail previously (5). The fractions containing
NADH-ferricyanide-reductase activity were collected, and the
fractions with the highest absorbance at 417 nm (cytochrome
b5) were combined.
amine was catalyzed mainly by the flavin-containing
monooxygenase (FMO) system (13, 14). Human FMO3
also N-oxygenates methamphetamine (14). Baba et al.
(12) showed that the P4501 system was also capable of
methamphetamine N-hydroxylation and isolated a P450
enzyme from rat liver that was predominantly involved
in the N-hydroxylation of methamphetamine.
For the metabolism of amphetamine by P450 and the
FMO, four general pathways are well-known, including
aromatic hydroxylation, aliphatic hydroxylation, oxida-
tive deamination, and N-oxidation (14-18).
During our investigations of the metabolism of guani-
dines, we observed an increase in the extent of P450-
mediated N-hydroxylation by adding of N-methylhy-
droxylamine to the incubation mixture (3). We postulated
that N-methylhydroxylamine (Scheme 1) inhibited the
retroreduction and increased the apparent rate of N-
hydroxylation to N-hydroxylated products. The reduction
of N-methylhydroxylamine by pig liver microsomes and
the hydroxylamine reductase has been described without
identifying all the components of the enzyme system (19).
The object of the study presented here was to deter-
mine if benzamidoxime reductase detoxicates N-hydroxy-
lated aliphatic amines by reducing the hydroxylamine to
the parent amine. We determined that the hydroxyl-
amine reductase and the benzamidoxime reductase were
identical. As model compounds, the hydroxylamines of
amphetamine, methamphetamine, and methylamine were
examined and found to be good substrates for this
reductase system.
Cytoch r om e b5. Cytochrome b5 was purified on a DEAE-
cellulose column as described previously (5). The final cyto-
chrome b5 fraction contained 17.22 nmol of cytochrome b5/mg
of protein.
Ben za m id oxim e Red u cta se. Benzamidoxime reductase
was chromatographed on an anion exchange column by pre-
parative HPLC with a conventional HPLC system (L-6210
Intelligent Pump, 655 A-22 UV detector, D-2500 integrator;
Merck/Hitachi, Darmstadt, Germany). Approximately 1 mg of
protein was applied to a semipreparative Fractogel EMD TMAE
650 (S) column (150 mm × 10 mm; particle size, 25-40 µm;
Merck) that was equilibrated with buffer A [10 mM Tris-acetate
(pH 7.4), 0.1 mM EDTA, 0.1 mM dithiothreitol, 20% (w/v)
glycerol, and 0.5% (w/v) Thesit]. After equilibration, fractions
were eluted with a one-step increase in the salt concentration
up to 0.5 M in buffer A at a flow rate of 1 mL/min. The first
fraction, which contained the benzamidoxime reductase, was
purified from UDP-glucuronyltransferase on an UDP-hexano-
lamine-sepharose (Sigma) column (4.5 cm × 1.2 cm) equilibrated
with 10 mM Tris-acetate buffer (pH 7.4) and washed with 50
mL of buffer B, consisting of 50 mM KCl and 40 µM phosphatid-
ylcholine in 10 mM Tris-acetate buffer (pH 7.4). The second
fraction contained the NADH-cytochrome b5-reductase.
NADH-Cytoch r om e b5-Red u cta se. NADH-cytochrome b5-
reductase was purified to homogeneity by affinity chromatog-
raphy on 5′-AMP-Sepharose 4B (Pharmacia) (22). The fractions
containing the highest NADH-ferricyanide-reductase activity
were combined and concentrated, followed by gel filtration (NAP
10, Pharmacia). The specific activity of the purified reductase
was 38.1 units/mg of protein.
Ma ter ia ls a n d Meth od s
Ch em icals. Methamphetamine and amphetamine were kindly
supplied by P. Ro¨sner (Landeskriminalamt Schleswig-Holstein,
Kiel, Germany) or the National Institute on Drug Abuse Drug
Supply Program (National Institutes of Health, Rockville, MD).
N-Methylhydroxylammonium chloride and NADH (disodium
salt) as well as all other chemicals and solvents were obtained
from E. Merck (Darmstadt, Germany). All chemicals were
analytical grade.
Detergents were removed from the purified enzymes by
shaking the concentrated fractions for 4 h with Calbiosorb
(Calbiochem, La J olla, CA) at 4 °C.
1 Abbreviations: P450, cytochrome P450; DEAE, diethylaminoethyl;
TMAE, trimethylaminoethyl; DLPC, dilaurylphosphatidylcholine.

Reduction of Aliphatic Hydroxylamines
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1039
An a lytica l P r oced u r es. (1) P r otein Con cen tr a tion . Pro-
tein concentrations were measured using the method described
by Smith et al. (23) with bicinchoninic acid (BCA reagent kit,
Pierce Chemical Co., Rockford, IL). All photometric measure-
ments were performed with a Uvicon 930 (Kontron Instruments,
Neufahrn, Germany) spectrophotometer.
(2) Cytoch r om e P 450 Con cen tr a tion s. The P450 concen-
tration was analyzed using the method of Omura and Sato (24).
(3) Cytoch r om e b5 Con cen tr a tion s. The cytochrome b5
concentration was determined by recording the reduced minus
the oxidized spectrum (absorbance at 185 mM-1 cm-1) as
described by Estabrook and Werringloer (25).
(4) NADH-Cytoch r om e b5-Red u cta se. NADH-cytochrome
b5-reductase was assessed by its NADH-ferricyanide-reductase
activity (1 unit ) 1 µmol of reduced ferricyanide/min) as
described by Mihara and Sato (26).
(5) Ben za m id oxim e Red u cta se. Benzamidoxime reductase
activity was measured by reduction of benzamidoxime to benz-
amidine and monitored by HPLC as described by Clement et
al. (5).
In cu ba tion s. (1) Red u ction of Meth a m p h eta m in e Hy-
d r oxyla m in e. Incubations were carried out in a shaking water
bath at 37 °C in the presence of oxygen using 1.5 mL reaction
vessels. The standard incubation mixture (150 µL) contained
the following components: 100 mM potassium phosphate buffer
(pH 6.3), 1.9 mM methamphetamine hydroxylamine, 3 µg of
benzamidoxime reductase, 0.4 unit of NADH-cytochrome b5-
reductase, 87 pmol of cytochrome b5, 40 mM DLPC (Sigma), and
2 mM NADH. After preincubation for 3 min at 37 °C, the
reactions were started by addition of NADH. The incubation
time was 30 min. The reaction was terminated by addition of
150 µL of methanol and by cooling the samples in ice. After
centrifugation at 10000g and 4 °C, 10 µL aliquots of the
supernatant were directly analyzed by HPLC.
(6) Micr osom a l Red u ction of N-Meth ylh yd r oxyla m in e.
Incubations were performed as described above but with addi-
tion of 30 µg of human liver microsomes instead of cytochrome
b5, NADH-cytochrome b5-reductase, DLPC, and benzamidoxime
reductase.
HP LC. (1) Meth a m p h eta m in e Hyd r oxyla m in e. The re-
sulting clear supernatants from incubations were analyzed using
high-performance liquid chromatography (616 HPLC pump and
600S controller from Waters, Milford, CT) equipped with a
variable-wavelength UV detector (Waters 486 TAD) set at 220
nm and an autosampler (Waters 717 plus WISP). The areas
under the peak were integrated with the EZChrom Chroma-
tography Data System (version 6.7, Scientific Software Inc., San
Ramon, CA). Separation and quantification were performed at
room temperature on an Aluspher RP-select B-column (244 mm
× 4 mm; particle size, 5 µm; Merck). The mobile phase was 0.025
M NaOH/methanol (70:30, v/v) and was passed through the
column at a rate of 1.0 mL/min. The injected sample volume
was 10 µL. Solvents used in the analysis were filtered through
a Sartolon membrane filter (0.45 µm, Sartorius AG, Goettingen,
Germany) and degassed by bubbling with helium or sonication.
For the determination of the recovery efficiency and the detec-
tion limit of the metabolite methamphetamine, known concen-
trations of the synthetic reference substance (from 14 to 90 µM)
dissolved in the mobile phase were measured. The standard
curves were linear over this range with correlation coefficients
of 0.998. The detection limit was about 5 µM which corresponded
to a rate of reduction of 1.17 nmol of methamphetamine min-1
(mg of benzamidoxime reductase)-1. The retention times were
5.8 ( 0.5 min for methamphetamine hydroxylamine and 7.6 (
0.5 min for methamphetamine.
(2) Am p h eta m in e Hyd r oxyla m in e. Separation and quan-
tification were carried out as described above for methamphet-
amine hydroxylamine. The mobile phase was 0.025 M NaOH/
methanol (90:10, v/v). Standard curves at the levels from 63 µM
to 0.71 mM amphetamine were constructed and found to be
linear over this range with correlation coefficients of 0.964. The
recovery rate of amphetamine from incubation mixtures without
adding cofactor was 91.5 ( 2.5% (N ) 32).
(2) Hu m a n Liver Micr osom a l Red u ction of Meth a m -
p h eta m in e Hyd r oxyla m in e. Incubations were carried out as
described above but with addition of 30 µg of human liver
microsomes instead of cytochrome b5, NADH-cytochrome b5-
reductase, DLPC, and benzamidoxime reductase.
The detection limit was about 57 µM, which corresponded to
a rate of reduction of 0.32 nmol of amphetamine min-1 (mg of
benzamidoxime reductase)-1. The retention times were 5.2 (
0.5 min for amphetamine hydroxylamine and 7.5 ( 0.5 min for
amphetamine.
(3) Red u ction of Am p h eta m in e Hyd r oxyla m in e. Incuba-
tions were carried out in a shaking water bath at 37 °C in the
presence of oxygen using 1.5 mL reaction vessels. The standard
incubation mixture (150 µL) contained the following compo-
nents: 100 mM potassium phosphate buffer (pH 6.3), 0.5 mM
amphetamine hydroxylamine, 2.5 µg of benzamidoxime reduc-
tase, 0.33 unit of NADH-cytochrome b5-reductase, 72.5 pmol of
cytochrome b5, 40 mM DLPC (Sigma), and 2 mM NADH. After
preincubation for 3 min at 37 °C, the reactions were started by
addition of NADH. The incubation time was 30 min. The
reaction was terminated by addition of 150 µL of methanol and
by cooling the samples in ice. After centrifugation at 10000g
and 4 °C, 10 µL aliquots of the supernatant were directly
analyzed by HPLC.
UV/Vis Sp ect r op h ot om et r y. N-Met h ylh yd r oxyla m in e.
The resulting clear supernatant was analyzed using the indirect
spectrophotometric analysis of N-methylhydroxylamine as de-
scribed by Kadlubar et al. (19). An aliquot of 250 µL of the clear
supernatant was diluted with ethanol to afford a volume of 450
µL. Sodium acetate buffer (200 µL, 1 mM, pH 4.6), 200 µL of a
10 mM ethanolic bathophenanthrolin solution, and 50 µL of a
10 mM ferrinitrate solution in sodium acetate buffer were added.
After exactly 3 min, 100 µL of a 10 mM EDTA solution in sodium
acetate buffer was added. Within 2 min, the solution was
assessed with a wavelength set at 535 nm and compared with
a solution of 50 mM Tris buffer (pH 6.3) and methanol (1:1, v/v)
instead of the aliquot. Standard curves in the range from 25
µM to 0.15 mM N-methylhydroxylamine were constructed and
found to be linear over this range with correlation coefficients
of 0.9954.
(4) Hu m a n Liver Micr osom a l Red u ction of Am p h et-
a m in e Hyd r oxyla m in e. Incubations were performed as de-
scribed above but with addition of 30 µg of human liver
microsomes instead of cytochrome b5, NADH-cytochrome b5-
reductase, DLPC, and benzamidoxime reductase.
(5) Red u ction of N-Meth ylh yd r oxyla m in e. Incubations
were carried out in a shaking water bath at 37 °C in the
presence of oxygen using 1.5 mL reaction vessels. The standard
incubation mixture (300 µL) contained the following compo-
nents: 50 mM Tris-acetate buffer (pH 6.3), 0.2 mM N-methyl-
hydroxylamine, 5 µg of benzamidoxime reductase, 0.5 unit of
NADH-cytochrome b5-reductase, 100 pmol of cytochrome b5, 40
mM DLPC (Sigma), 500 units/mL superoxide dismutase, and 1
mM NADH. After preincubation for 2 min at 37 °C, the reactions
were started by addition of NADH. The incubation time was 15
min. The reaction was terminated by the addition of 300 µL of
methanol and cooling the samples in ice. After centrifugation
at 10000g and 4 °C, the disappearance of the hydroxylamine
was assessed by UV/vis spectrophotometry.
Neu r on a l Cell Cu ltu r e a n d Am p h eta m in e a n d Meth -
a m p h eta m in e Hyd r oxyla m in e Toxicity. Primary neuronal
material was obtained from two 15-17-day-old rat embryos.
Appropriate approval from the institutional animal care and
use committee was obtained for this study. The pregnant rats
were anesthetized with ether, and the embryos were removed.
The whole brain was next removed with forceps, and the brain
was dissected. Because of the ease and yield of cell culturing,
the studies were carried out with cerebral cortex. After removal
of the thalmus, the cerebral cortex was dispersed and passed
through 80 µm nylon mesh into Eagle’s modified essential
medium containing 30 mM glucose and 5 µg of insulin/mL,

1040 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Clement et al.
Ta ble 1. In Vitr o Red u ction of Meth a m p h eta m in e Hyd r oxyla m in e to Meth a m p h eta m in e by Ben za m id oxim e Red u cta se
a n d Hu m a n a n d P ig Liver Micr osom esa
nmol of methamphetamine
preparation
composition
complete mixture
Nb
min-1 (mg of protein)-1
benzamidoxime reductase
8
4
4
4
8
4
4
4
8
4
4
4
220.6 ( 27.2
7.3 ( 2.6
8.4 ( 1.2
NDc
11.0 ( 0.4
1.9 ( 0.2
2.0 ( 0.3
NDc
7.3 ( 0.5
1.2 ( 0.5
1.3 ( 0.3
NDc
without NADH
without benzamidoxime reductase
without methamphetamine hydroxylamine
complete mixture
without NADH
without microsomes
without methamphetamine hydroxylamine
complete mixture
without NADH
without microsomes
without methamphetamine hydroxylamine
pig liver microsomes
human liver microsomes
a
A complete incubation mixture for optimized conditions consisted of 1.9 mM methamphetamine hydroxylamine, 3 µg of benzamidoxime
reductase, 0.4 unit of NADH-cytochrome b5-reductase, 87 pmol of cytochrome b5, 40 mM DLPC (Sigma), and 2 mM NADH, in 150 µL of
100 mM potassium phosphate buffer (pH 6.3). Incubation mixtures with microsomes consisted of 30 µg of microsomal protein, as described
b
in Materials and Methods. Data are means ( the standard error from N different determinations; p < 0.05. Number of determinations.
c Not detected.
F igu r e 1. Representative HPLC chromatogram of the incubation of methamphetamine hydroxylamine with benzamidoxime
reductase. The incubation mixtures were as described in Materials and Methods: (a) complete incubation mixture and (b) incubation
mixture without NADH.
250 000 IU/L penicillin, 0.5% streptomycin, and 20% fetal calf
serum (v/v) supplemented with extra agents (27). The cells were
maintained in a humidified atmosphere. The NUNC A/S Petri
dishes that were used were precoated with sterile poly(L-lysine)
in boric acid/NaOH at pH 8.4. Neuronal cells were initially
plated at a density of 0.1-0.5 million cells/mL in polylysinated
12-well plates. After cultivation for 1 day, the medium was
changed, and after 4-5 days, the fetal calf serum level was
changed from 20 to 10%. Also, after 4-5 days, Ara-c (10-5 M)
was added to suppress the growth of astroglia (28). Using this
procedure, about 80-85% of the amount of neurons in culture
from prenatal brain were obtained (29, 30). The cells were
characterized with respect to cellular content by morphological
inspection. The cell viability of isolated neurons was checked
by lactate dehydrogenase release and trypan blue exclusion.
that of the synthetic material. Addition of the reference
substrate to the incubation mixture gave rise to an
increase in the area of the metabolite peak. This was also
reproduced when the HPLC eluent was varied (data not
shown). The chromatogram of the incubation mixture in
the absence of cofactor (NADH) (Figure 1) exhibited a
smaller peak for methamphetamine. The apparent re-
ductase activity in control incubations without NADH
resulted from impurities of the substrate methamphet-
amine hydroxylamine with methamphetamine and from
chemical reduction under the conditions of the incubation
(data not shown). For incubation times of 30 min, the
enzymatic reduction of methamphetamine hydroxyl-
amine proceeded linearly (data not shown).
The species dependency (Table 1) revealed that hy-
droxylamine reduction was more pronounced in pig liver
while appreciably lower conversion rates were observed
in human liver preparations.
The reduction of methamphetamine hydroxylamine in
completely reconstituted systems and NADH obeyed
Michaelis-Menten kinetics. The kinetic data are sum-
marized in Table 2.
Resu lts
Red u ction of Meth a m p h eta m in e Hyd r oxyla m in e.
The relative rates of in vitro reduction of methamphet-
amine hydroxylamine to methamphetamine by the benz-
amidoxime reductase and by microsomal fractions from
pig and human livers are listed in Table 1. A representa-
tive HPLC chromatogram recorded after the incubation
of methamphetamine hydroxylamine with the benzami-
doxime reductase shown in Figure 1 showed that the
retention time for the metabolite (7.6 min) agreed with
Red u ction of Am p h eta m in e Hyd r oxyla m in e. The
reduction of amphetamine hydroxylamine to amphet-
amine in vitro by the highly purified benzamidoxime

Reduction of Aliphatic Hydroxylamines
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1041
Ta ble 2. Kin etic Da ta for th e Red u ction of Meth a m p h eta m in e Hyd r oxyla m in e, Am p h eta m in e Hyd r oxyla m in e, a n d
N-Meth ylh yd r oxyla m in e by Ben za m id oxim e Red u cta se a n d Hu m a n Liver Micr osom es
Vmax [nmol min-1
Vmax/Km [L min-1
Km (mM)
(mg of protein)-1
]
(mg of protein)-1
]
methamphetamine hydroxylamine by benzamidoxime reductase
amphetamine hydroxylamine by benzamidoxime reductase
amphetamine hydroxylamine by human liver microsomes
N-methylhydroxylamine by benzamidoxime reductase (first phase)
N-methylhydroxylamine by benzamidoxime reductase (second phase)
5.32
0.49
0.76
0.21
0.57
193.3
4.37
4.01
60.24
722.2
3.63 × 10-5
8.92 × 10-6
5.26 × 10-6
2.87 × 10-4
1.27 × 10-3
Ta ble 3. In Vitr o Red u ction of Am p h eta m in e Hyd r oxyla m in e to Am p h eta m in e by Ben za m id oxim e Red u cta se a n d Hu m a n
a n d P ig Liver Micr osom esa
preparation
composition
complete mixture
Nb
nmol of amphetamine min-1 (mg of protein)-1
benzamidoxime reductase
8
4
4
4
8
4
4
4
8
4
4
4
5.25 ( 0.09
0.8 ( 0.31
NDc
without NADH
without benzamidoxine reductase
without amphetamine hydroxylamine
complete mixture
without NADH
without microsomes
without amphetamine hydroxylamine
complete mixture
without NADH
without microsomes
without amphetamine hydroxylamine
NDc
pig liver microsomes
3.01 ( 0.42
1.41 ( 0.02
1.0 ( 0.30
NDc
4.09 ( 0.11
2.0 ( 0.06
1.98 ( 0.29
NDc
human liver microsomes
a
A complete incubation mixture for optimized conditions consisted of 0.5 mM amphetamine hydroxylamine, 2.5 µg of benzamidoxime
reductase, 0.33 unit of NADH-cytochrome b5-reductase, 72.5 pmol of cytochrome b5, 40 mM DLPC (Sigma), and 2 mM NADH, in 150 µL
of 100 mM potassium phosphate buffer (pH 6.3). Incubation mixtures with microsomes consisted of 30 µg of microsomal protein, as described
b
in Materials and Methods. Data are means ( the standard error from N different determinations; p < 0.05. Number of determinations.
c Not detected.
F igu r e 2. Representative HPLC chromatogram of the incubation of amphetamine hydroxylamine with benzamidoxime reductase.
The incubation mixtures were as described in Materials and Methods: (a) complete incubation mixture and (b) incubation mixture
without NADH.
reductase and by microsomal fractions from pig and
human livers has been demonstrated (Table 3). A rep-
resentative HPLC chromatogram recorded after the
incubation of amphetamine hydroxylamine with the
benzamidoxime reductase is shown in Figure 2. The
retention time for the metabolite (7.5 min) agreed with
that of the authentic synthetic material. Addition of the
reference substrate to the incubation mixture gave rise
to an increase in the area of the metabolite peak when
the HPLC eluent was varied (data not shown). The
chromatogram of the incubation mixture in the absence
of cofactor (NADH) (Figure 2) exhibited a smaller peak
for amphetamine. The apparent reductase activity in
control incubations without NADH resulted from impuri-
ties of the substrate amphetamine hydroxylamine with
amphetamine and from chemical reduction under the
conditions of the incubation (data not shown). It is known
that in particular primary hydroxylamines are usually
quite unstable compounds. Thus, the chemical formation
of amphetamine could not be avoided completely. For
incubation times of 45 min, the reaction proceeded
linearly and exhibited a pH optimum at 5.1 (data not
shown).
The reduction of amphetamine hydroxylamine in com-
pletely reconstituted systems and NADH obeyed Michae-
lis-Menten kinetics. The kinetic data are summarized
in Table 2.
The reduction of amphetamine hydroxylamine in com-
plete systems with human liver microsomes and NADH
also obeyed Michaelis-Menten kinetics (Table 2).
The species dependency (Table 3) revealed that am-
phetamine hydroxylamine reduction was more pro-
nounced in human liver microsomes while appreciably
lower conversion rates were observed in pig liver mi-

1042 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Clement et al.
Ta ble 4. In Vitr o Red u ction of N-Meth ylh yd r oxyla m in e to N-Meth yla m in e by Ben za m id oxim e Red u cta se a n d Hu m a n
Liver Micr osom esa
ratio of N-reduction
of N-methylhydroxylamine
preparation
composition
complete mixture
Nb
[nmol min-1 (mg of protein)-1
]
benzamidoxime reductase
8
4
4
4
8
4
4
4
153 ( 15
NDc
without NADH
without benzamidoxine reductase
without N-methylhydroxylamine
complete mixture
without NADH
without microsomes
NDc
NDc
human liver microsomes
24.7 ( 1.71
2.0 ( 0.03
NDc
without N-methylhydroxylamine
NDc
a
A complete incubation mixture for optimized conditions consisted of 0.2 mM N-methylhydroxylamine, 5 µg of benzamidoxime reductase,
0.5 unit of NADH-cytochrome b5-reductase, 100 pmol of cytochrome b5, 40 mM DLPC (Sigma), 1 mM NADH, and 500 units/mL superoxide
dismutase, in 150 µL of 100 mM potassium phosphate buffer (pH 6.3). Incubation mixtures with microsomes consisted of 30 µg of microsomal
b
protein, as described in Materials and Methods. Data are means ( the standard error from N different determinations; p < 0.05. Number
of determinations. c Not detected.
F igu r e 4. Effect of different concentrations of N-methylhy-
droxylamine on the reduction of benzamidoxime to benzamidine
by benzamidoxime reductase. The incubation mixtures were as
described in Materials and Methods in the presence of different
concentrations of N-methylhydroxylamine. The amount of benz-
amidine in the absence of N-methylhydroxylamine under control
F igu r e 3. Effect of different concentrations of N-methylhy-
conditions was taken to be 100%. Data are means ( the
droxylamine on the reduction of amphetamine hydroxylamine
by benzamidoxime reductase. The incubation mixtures were as
described in Materials and Methods in the presence of different
concentrations of N-methylhydroxylamine. The amount of am-
phetamine in the absence of N-methylhydroxylamine under
control conditions was taken to be 100%. Data are means ( the
standard error from four determinations.
standard error from four determinations.
The effect of increasing concentrations of benzamidoxime
for the N-methylhydroxylamine reduction and the sub-
strate dependency are shown in Figures 6 and 7, respec-
tively. Kinetic data are summarized in Table 2.
Neu r on a l Cell Cu ltu r e a n d Am p h eta m in e a n d
Meth a m p h eta m in e Hyd r oxyla m in e Toxicity. Young
(i.e., 5-day-old) cultures containing astrocytes, oligoden-
drocytes, and microglia were incubated in the presence
of test compounds for 16 h. Panel A of Figure 8 shows
cells sham-treated with vehicle (i.e., ethanol). Panel B
shows cell cultures treated with 0.6 mM amphetamine
hydroxylamine and panel D cell cultures treated with 0.6
mM methamphetamine hydroxylamine. Although the
primary cultures were not fully differentiated, the data
show a striking effect for the compounds that were
administered. At 0.6 mM, both amphetamine hydroxyl-
amine and methamphetamine hydroxylamine caused
frank cell toxicity. Panels C and D of Figure 8 show
massive cell death and cytotoxicity.
crosomes. The effect of increasing concentrations of
N-methylhydroxylamine in the incubation mixtures is
shown in Figure 3.
Red u ction of N-Meth ylh yd r oxyla m in e. The reduc-
tion of N-methylhydroxylamine to methylamine in vitro
by the highly purified benzamidoxime reductase and by
microsomal fractions from pig liver is described in Table
4.
In incubation mixtures in the absence of cofactor
(NADH) or benzamidoxime reductase, no reduction of
N-methylhydroxylamine could be detected. For incuba-
tion times of 45 min, the reaction proceeded linearly.
The reduction of N-methylhydroxylamine in completely
reconstituted systems and NADH exhibited a biphasic
course. The kinetic data are summarized in Table 2.
The species dependency (Table 4) revealed that N-
methylhydroxylamine reduction was more pronounced in
humans while appreciably lower conversion rates were
observed in pig liver microsomes (19). The effect of
increasing concentrations of N-methylhydroxylamine in
the incubation mixtures of benzamidoxime reduction is
shown in Figure 4. The substrate dependency for N-
methylhydroxylamine reduction is shown in Figure 5.
At a comparable dose and incubation time, amphet-
amine and methamphetamine showed some detectable
cell toxicity but considerably less than their correspond-
ing hydroxylamines (data not shown). Amphetamine
oxime was virtually nontoxic at doses that far exceeded
that of the hydroxylamines. Compared with healthy
control cells (panel A), cells treated with 1.0 mM am-
phetamine oxime did not show detectable cell toxicity.

Reduction of Aliphatic Hydroxylamines
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1043
F igu r e 5. Direct plot of the reduction of benzamidoxime to
benzamidine by benzamidoxime reductase and its dependence
on different concentrations of N-methylhydroxylamine. The
incubation mixtures were as described in Materials and Methods
in the presence of different concentrations of N-methylhydroxyl-
amine. Data are means ( the standard error from four deter-
minations: ([) no N-methylhydroxylamine, (b) 50 µM N-
methylhydroxylamine, (9) 100 µM N-methylhydroxylamine, (2)
150 µM N-methylhydroxylamine, and (1) 200 µM N-methyl-
hydroxylamine.
F igu r e 7. Direct plot of the reduction of N-methylhydroxyl-
amine by benzamidoxime reductase and its dependence on
different concentrations of benzamidoxime. The incubation
mixtures were as described in Materials and Methods in the
presence of different concentrations of benzamidoxime. Data are
means ( the standard error from four determinations: (b) no
benzamidoxime, (O) 0.02 mM benzamidoxime, (9) 0.075 mM
benzamidoxime, (0) 0.2 mM benzamidoxime, (2) 0.5 mM benz-
amidoxime, and (1) 1.0 mM benzamidoxime.
Methamphetamine and amphetamine have a high
potential for abuse and a significant human toxic poten-
tial. The relative rate of hydroxylamine metabolite
formation and the relative rate of hydroxylamine reduc-
tion to parent amine may be important in the overall
toxicity of these compounds (31).
While limited, the effect of amphetamine hydroxyl-
amine and methamphetamine hydroxylamine on primary
rat brain cell cultures clearly shows that these metabo-
lites are cytotoxic at the concentrations that were used
(Figure 8). At the concentrations that were studied, it
appeared that methamphetamine hydroxylamine showed
greater cell toxicity than amphetamine hydroxylamine.
However, the kinetics of the onset of cell toxicity for each
compound may be different, and additional studies are
needed to clarify this point. It is notable that amphet-
amine oxime is virtually nontoxic at the dose that was
examined. This result is in keeping with the hypothesis
that oxime formation is a metabolic detoxication path-
way, converting toxic hydroxylamines into nontoxic oximes
(14). At comparable doses, the parent compounds am-
phetamine and methamphetamine showed significantly
less cytotoxicity. Thus, retroreduction of amphetamine
hydroxylamine or methamphetamine hydroxylamine to
the amine also represents a detoxication pathway.
The formation of the amines from the reactive metabo-
lites of methamphetamine hydroxylamine and amphet-
amine hydroxylamine was confirmed in each case by
means of an HPLC system coupled to a UV detector
(Figures 1 and 2). The reduction of N-methylhydroxy-
lamine was analyzed using an indirect spectrophotomet-
ric analysis (19).
F igu r e 6. Effect of different concentrations of benzamidoxime
on the reduction of N-methylhydroxylamine by benzamidoxime
reductase. The incubation mixtures were as described in
Materials and Methods in the presence of different concentra-
tions of benzamidoxime. The rate of N-methylhydroxylamine
conversion in the absence of benzamidoxime under control
conditions was taken to be 100%. Data are means ( the
standard error from four determinations.
Discu ssion
The objective of this study was to investigate the
reduction of aliphatic hydroxylamines to their parent
amines by the hepatic benzamidoxime reductase system.
On the basis of previous investigations (5), the enzyme
system is composed of cytochrome b5, NADH-cytochrome
b5-reductase, and the benzamidoxime reductase. For the
reduction of benzamidoxime to benzamidine (5), NADH
is preferred over NADPH as a cofactor, so the same
cofactor was used in the work presented here. The
reductions of methamphetamine hydroxylamine, am-
phetamine hydroxylamine, and N-methylhydroxylamine
have been carried out for the first time by this enzyme
system.
Methamphetamine hydroxylamine, amphetamine hy-
droxylamine, and N-methylhydroxylamine were also
reduced by liver microsomes from pigs and humans
(Tables 1, 3, and 4). Methamphetamine hydroxylamine
was efficiently metabolized by pig liver microsomes, but
amphetamine hydroxylamine is more efficiently reduced
by human liver microsomes than by pig liver microsomes.
In the presence of pig liver microsomes, Kadlubar et al.

1044 Chem. Res. Toxicol., Vol. 13, No. 10, 2000
Clement et al.
F igu r e 8. Effect of 0.6 mM amphetamine hydroxylamine (C) or 0.6 mM methamphetamine hydroxylamine (D) on primary rat
brain cell cultures. Compared to vehicle-treated control cells (A), cells treated with amphetamine hydroxylamine and methamphetamine
hydroxylamine showed massive cell toxicity and cell death. Amphetamine oxime administered to cell cultures at a concentration of
1.0 mM did not show detectable cell toxicity (B).
(19) observed a rate of reduction of 16.0 ( 0.5 nmol min-1
(mg of protein)-1 for N-methylhydroxylamine. We con-
clude that N-methylhydroxylamine is more efficiently
reduced by human liver microsomes than by pig liver
microsomes.
The addition of N-methylhydroxylamine in increasing
concentrations to incubation mixtures of amphetamine
hydroxylamine (Figure 3) and benzamidoxime (Figure 4)
effected a corresponding decrease in the rates of conver-
sion. In accordance with studies with N-hydroxydebriso-
quine (3) and guanoxabenz (4), the presence of N-meth-
ylhydroxylamine probably causes inhibition of these
reductions.
The Vmax/Km ratios for the reduction of amphetamine
hydroxylamine by the highly purified benzamidoxime
reductase and human liver microsomes (Table 2) were
lower than the Vmax/Km value for the reduction of benz-
amidoxime to benzamidine [Vmax/Km ) 5.3 × 10-4 L min-1
(mg of bezamidoxime reductase)-1] in the same systems
(5).
The kinetic data for the reduction of benzamidoxime
to benzamidine in the presence of increasing concentra-
tions of N-methylhydroxylamine (Figure 5) and the
resulting Kiu value of 91.7 µM and the Kic value of 61.1
µM showed all the characteristics of a mixed noncompeti-
tive enzyme inhibition.
The ratios of the reduction by benzamidoxime reduc-
tase were significantly higher than the value observed
for the reduction by human liver microsomes. The values
for the reduction of N-methylhydroxylamine were in the
same range as those for the reduction of benzamidoxime
by benzamidoxime reductase (5). In comparison, the level
of reduction of benzamidoxime (5) was 140% of that of
methamphetamine hydroxylamine. In contrast, the level
of reduction of amphetamine hydroxylamine was only
about 4% of that of benzamidoxime.
The kinetic data for the reduction of N-methylhydroxyl-
amine in the presence of increasing concentrations of
benzamidoxime (Figure 7) and the resulting K values
(Kiu1 ) 102.8 µM, Kic1 ) 6.94 mM, Kiu2 ) 16.9 µM, and
Kic2 ) 49.61 µM) also showed the characteristics of a
mixed noncompetitive enzyme inhibition.
According to Kadlubar and Ziegler (7), the enzyme
system consists of cytochrome b5, NADH-cytochrome b5-
reductase, and a third unidentified protein. We isolated
a P450 2D enzyme from pig liver which in combination
with cytochrome b5 and its reductase reduced benzami-
doxime very efficently (5) and as shown here also reduces
aliphatic hydroxylamines.
The Vmax/Km value for the reduction of amphetamine
hydroxylamine by benzamidoxime reductase was higher
than that for the reduction by human liver microsomes
(Table 2). The value for the reduction of amphetamine
hydroxylamine was significantly lower than that for the
reduction of methamphetamine hydroxylamine. Although
both Vmax/Km values were lower than the ratio for the
The question arose as to which P450 enzyme is
involved in the hydroxylamine reduction. In pig liver, it
is P450 2D (5). However, human microsomes from donors
deficient in 2D6 were also able to reduce benzamidoxime
(5). Studies with inhibitors did not give a hint as to which
human P450 enzyme is involved. The reduction of benz-
reduction of benzamidoxime [Vmax/Km ) 5.3 × 10-4
L
min-1 (mg of benzamidoxime reductase)-1] (5), they were
comparable with those of other enzymatic reductions,
including that with xanthine oxidase (32).

Reduction of Aliphatic Hydroxylamines
Chem. Res. Toxicol., Vol. 13, No. 10, 2000 1045
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Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft and the Fonds der
Chemischen Industrie. The financial assistance of the
National Institutes of Health (Grants GM36246 and
DA11547) is gratefully acknowledged. We are indebted
to Monika Henke, Meike Wollny, and Sven Wichmann
for their excellent technical assistance, to Dr. Lifen Xu
for the synthesis of amphetamine hydroxylamine and
methamphetamine hydroxylamine, and to Dr. J ohn
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