Enzyme induction and comparative oxidative desulfuration of isothiocyanates to isocyanates

Article abstract of DOI:10.1021/tx950213f

Enzyme induction of oxidative metabolism of isothiocyanates to isocyanates by rat liver microsomes and comparative metabolic conversion of some isothiocyanates were investigated. Metabolic activity was assayed by trapping the isocyanate metabolites from isothiocyanates with the inclusion of 2-aminofluorene to form the respective mixed ureas as previously described for the 2-naphthyl isothiocyanate. Male F344 rats were fed either a conventional grain diet for induction with Aroclor 1254 or AIN 76A diet without antioxidant beginning 2 weeks before treatment with Aroclor 1254, β- naphthoflavone, isosafrole, or phenobarbital. Enzymes responsible for the metabolism of 1- and 2-naphthyl isothiocyanate were inducible by all four agents, Aroclor being the best under the current induction protocol and metabolic conversion assay procedure. On the other hand, enzymes responsible for the metabolism of benzyl isothiocyanate were induced only by Aroclor and, to a lesser extent, by phenobarbital. For the comparative metabolic conversion studies, using the microsomes from Aroclor-treated rats fed a conventional grain diet, the rates of metabolic conversion followed the order of 1-naphthyl >> phenyl > benzyl and phenethyl >> propyl, ethyl, and methyl isothiocyanates.

Full text of DOI:10.1021/tx950213f

1072
Chem. Res. Toxicol. 1996, 9, 1072-1078
En zym e In d u ction a n d Com p a r a tive Oxid a tive
Desu lfu r a tion of Isoth iocya n a tes to Isocya n a tes
Mei-Sie Lee
Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 East
Warren Avenue, Detroit, Michigan 48201
Received December 21, 1995X
Enzyme induction of oxidative metabolism of isothiocyanates to isocyanates by rat liver
microsomes and comparative metabolic conversion of some isothiocyanates were investigated.
Metabolic activity was assayed by trapping the isocyanate metabolites from isothiocyanates
with the inclusion of 2-aminofluorene to form the respective mixed ureas as previously described
for the 2-naphthyl isothiocyanate. Male F344 rats were fed either a conventional grain diet
for induction with Aroclor 1254 or AIN 76A diet without antioxidant beginning 2 weeks before
treatment with Aroclor 1254, â-naphthoflavone, isosafrole, or phenobarbital. Enzymes
responsible for the metabolism of 1- and 2-naphthyl isothiocyanate were inducible by all four
agents, Aroclor being the best under the current induction protocol and metabolic conversion
assay procedure. On the other hand, enzymes responsible for the metabolism of benzyl
isothiocyanate were induced only by Aroclor and, to a lesser extent, by phenobarbital. For
the comparative metabolic conversion studies, using the microsomes from Aroclor-treated rats
fed a conventional grain diet, the rates of metabolic conversion followed the order of 1-naphthyl
. phenyl > benzyl and phenethyl . propyl, ethyl, and methyl isothiocyanates.
cytochrome P450 system, and the enzymes responsible
for this conversion were inducible by Aroclor 1254.
Further studies have shown that 1-naphthyl isothio-
cyanate (1-NITC) and dietary benzyl isothiocyanate
(BITC) can also be metabolized to reactive isocyanates
(30). Although both isomers of naphthyl isothiocyanate
were metabolized to a comparable extent by the Aroclor-
inducible P450(s), the metabolism of BITC was much
lower and Aroclor treatment of rats fed the conventional
grain diet did not increase the enzyme activity. The
latter observations prompted studies of enzyme induction
with three additional common inducers, i.e., â-naphtho-
flavone, isosafrole, and phenobarbital, to gain insight into
the enzymes that are responsible for the metabolic
conversion of these compounds. Animals were fed AIN
76A diet without ethoxyquin prior to and during the
treatment with the inducing agents in an attempt to
minimize the effects of induction due to dietary compo-
nents, including ethoxyquin which is known to induce
P450 2B1, 2B2, 1A2, and 3A2 (31).
In tr od u ction
Organic isocyanates (RNdCdO) are used extensively
in industries, and production of these compounds is on
the rise in the United States (1). Apart from direct
occupational exposure, exposures can also come from
chemotherapeutic dialkylnitrosoureas (2) and industrial
solvents such as alkylformamides which can be metabo-
lized to isocyanates (3). Since isocyanates are reactive
electrophilic agents, they are capable of modifying pro-
teins (4) and nucleic acids in vitro (5, 6) and in vivo (7);
biologically, they can cause chromosome aberrations,
sister-chromatid exchanges (8-11), mutations (6, 8, 12,
13) and cancer (14, 15).
Organic isothiocyanates (RNdCdS), the sulfur analogs
of isocyanates, possess a wide range of biological activi-
ties, and hence, they have been used medicinally (16).
Moreover, isothiocyanates with diverse structural fea-
tures can be generated from a variety of vegetables by
enzymatic hydrolysis of their glucosinolates with my-
rosinase (17). Several synthetic and dietary isothio-
cyanates have been used as chemopreventive agents in
experimental animals (18-25) and have been considered
for use in humans. A number of isothiocyanates have
been shown to be mutagenic for Salmonella typhimurium
TA100 (26, 27), and mutagenic dietary allyl isothiocyan-
ate has been found to cause bladder tumors in F344 rats
(28).
Metabolic conversion was further studied with aryl,
arylalkyl, and aliphatic isothiocyanates using Aroclor-
induced liver microsomes.
Ma ter ia ls a n d Meth od s
Ca u tion : Aromatic amines, isocyanates, isothiocyanates, and
Aroclor 1254 are hazardous and should be handled with care.
Ma ter ia ls. Aroclor 1254 was a gift from Dr. P. Hollenberg,
Department of Pharmacology, University of Michigan School of
Recently, it has been found that reactive isocyanates
such as 2-naphthyl isocyanate (6) and methyl isocyanate
(5) formed predominantly N4-cytosine adducts and ex-
erted mutagenic effects in a bacteriophage system (6, 12).
Since we are likely to be exposed to isothiocyanates, it is
of interest that their genotoxic effects be evaluated. The
results of such an effort have been reported (29), in which
2-naphthyl isothiocyanate (2-NITC)1 was converted to the
more reactive and toxic 2-naphthyl isocyanate by a
1
Abbreviations: 2-AF, 2-aminofluorene; BFU, N-benzyl-N′-(2-fluo-
renyl)urea; BITC, benzyl isothiocyanate; EFU, N-ethyl-N′-(2-fluorenyl)-
urea; EITC, ethyl isothiocyanate; MFU, N-methyl-N′-(2-fluorenyl)urea;
MITC, methyl isothiocyanate; 1-NFU, N-(1-naphthyl)-N′-(2-fluorenyl)-
urea; 1-NITC, 1-naphthyl isothiocyanate; 2-NFU, N-(2-naphthyl)-N′-
(2-fluorenyl)urea; 2-NITC, 2-naphthyl isothiocyanate; PEFU; N-phen-
ethyl-N′-(2-fluorenyl)urea; PEITC, phenethyl isothiocyanate; PFU,
N-phenyl-N′-(2-fluorenyl)urea; PITC, phenyl isothiocyanate; PrFU,
N-propyl-N′-(2-fluorenyl)urea; PrITC, propyl isothiocyanate; P450,
cytochrome P450.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0893-228x(95)00213-X CCC: $12.00 © 1996 American Chemical Society

Oxidative Desulfuration of Isothiocyanates
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1073
MA) were used in this study. The animals were fed either a
conventional grain diet (Harlan Teklad 8604, Armada Grain Co.,
Bartonville, IL) for induction with Aroclor 1254 as described
previously (29) or the semisynthetic AIN 76A diet without
antioxidant (United States Biochemical Corp., Cleveland, OH)
starting 2 weeks prior to the first injections for the comparative
induction studies with Aroclor and three additional inducers.
These compounds were dissolved or suspended in trioctanoin
in the following doses (mg inducer/1.3 mL of trioctanoin/kg body
weight): Aroclor 1254, 500 (single injection followed by 3
injections with solvent); â-naphthoflavone, 80; isosafrole, 150;
sodium phenobarbital, 60. With the exception of Aroclor, the
animals in groups of 3-4 were injected once daily for 4 days,
and control animals received 4 daily injections with trioctanoin
alone. On day 5, after overnight starvation, the animals were
sacrificed and the liver microsomes were prepared according to
the published procedure (29), except that the liver homogenates
were centrifuged at 9000g for 30 min at 4 °C before the
supernatants were centrifuged at 105000g. For comparative
metabolic conversion studies, the pooled microsomes from 6 of
Aroclor-treated animals fed conventional diet were used. The
protein content was determined by the method of Bradford (37),
and the microsomes were stored in small aliquots at -80 °C
until use.
Sch em e 1. Sch em e for th e Meta bolic Con ver sion of
Isoth iocya n a tes a n d Tr a p p in g of th e Rea ctive
Isocya n a te Meta bolites w ith 2-AF
Medicine, Ann Arbor, MI 48109. Trioctanoin was from Pfaltz
and Bauer (Stamford, CT). Sodium phenobarbital and NADPH
(type III) were from Sigma Chemical Co. (St. Louis, MO). All
other chemicals used in this study were either purchased from
Aldrich Chemical Co. (Milwaukee, WI) or synthesized in this
laboratory.
Meta bolic Con ver sion Assa ys. Analyses of the metabolic
conversion of isothiocyanates to isocyanates were carried out
in the presence of 2-AF to trap the reactive metabolites as mixed
ureas RNHCONHF (III) as described previously for 2-NITC (29).
Briefly, for comparative induction studies, 1 mL of the complete
incubation mixture contained 0.4 mg of microsomal protein, 0.5
µmol of substrate (2-NITC, 1-NITC, and BITC)/10 µL of DMSO,
5 µmol of NADPH and 0.2 µmol of 2-AF/10 µL of DMSO. All
incubations were at 37 °C for 30 min which had produced a time-
dependent yield of products when 2-NITC was the substrate
(29). For comparative metabolic conversion studies, incubation
mixtures were proportionally increased to 2 mL to contain 1.0
µmol of substrate [1-NITC, PITC, PrITC (synthetic), BITC,
PEITC, EITC, and MITC (naturally occurring)]/20 µL of DMSO.
The time course was from 0 up to 60 min. Ethyl acetate extracts
of the incubation mixtures (except for EITC, see further treat-
ment below) were evaporated to dryness and redissolved in
methanol or acetonitrile containing 10% dimethylformamide for
analyses by reverse-phase HPLC. For analysis of EITC me-
tabolite, in order to avoid the interference of product quantita-
tion due to the large neighboring 2-AF peak, the residues were
dissolved in 400 µL of acetone and incubated at 45 °C for 1 day
with 40 µL of acetone solution of 20 µmol of succinic anhydride
containing 10% of triethylamine. The carboxyl-containing
amide resulted from the reaction of succinic anhydride with
2-AF was removed by extraction of the ethyl acetate solution
with 0.2 N NaOH (2 × 500 µL) and then with water twice. The
residue thus obtained was subjected to HPLC analysis.
In str u m en ta tion a n d Gen er a l P r oced u r es for Ch em ica l
An a lysis. UV spectra were recorded using a Hewlett Packard
8452A diode array spectrophotometer. 1H-NMR spectra were
recorded with a GE NMR QE-300 instrument. Mass spectra
were recorded using a Kratos MS80RFA mass spectrometer.
Progress of reactions and purities of compounds were checked
by TLC using Whatman PE SIL G/UV silica gel polyester sheets
with benzene/95% ethanol ) 20/1 or 9/1 as solvent, and
chromatograms were viewed under UV light. Reverse-phase
HPLC analyses were carried out using a Waters 486 tunable
absorbance detector interfaced with a Nelson Model Turbo
Chrom Chromatography Software. An LKB Model 2140 diode
array detector was used as an in-line device for compound
identification. µBondapak C18 columns maintained at 35 °C
were used throughout the study.
Ch em ica l Syn t h eses. Di-2-naphthylurea, di-2-naphthyl-
thiourea, N-(2-naphthyl)-N′-(2-fluorenyl)urea (2NFU), 2-NITC,
and N-(2-naphthyl)-N′-(2-fluorenyl)thiourea were prepared as
described previously (29). Following similar procedures, all four
groups of compounds as depicted in Scheme 1 were either
purchased from Aldrich or prepared by reacting isocyanates or
isothiocyanates (R ) 1-naphthyl, phenyl, benzyl, phenethyl,
propyl, ethyl, and methyl) with their respective amines or
2-aminofluorene (2-AF) in inert solvents such as dry ether,
acetone, or dimethylformamide. The melting points of the
synthetic compounds are listed below. Symmetrically disub-
stituted ureas, RNHCONHR (I): R, 1-naphthyl, 297 °C, [lit. mp
296 °C (32)]; benzyl, 168-170 °C [lit. mp 169-170 °C (33)];
phenethyl, 137-139 °C [lit. mp 138 °C (34)]; propyl, 104-106
°C [lit. mp 105 °C (34)]. Symmetrically disubstituted thioureas,
RNHCSNHR (II): R, 1-naphthyl, 208-209 °C [lit. mp 207-208
°C (32)]; benzyl, 146-147 °C [lit. mp 147-148 °C (33)]; phen-
ethyl, 94-95 °C [lit. mp 94-95 °C (35)]; propyl, 71-72 °C [lit.
mp 72 °C (36)]. N-Substituted-N′-(2-fluorenyl)urea, RNH-
CONHF (III): R, 1-naphthyl, 263-278 °C; phenyl, ∼310 °C dec;
benzyl, 239-240 °C; phenethyl, 222-223 °C; propyl, 208-209
°C; ethyl, 236-238-310 °C dec; methyl, 219-221 °C. N-Sub-
stituted-N′-(2-fluorenyl)thiourea, RNHCSNHF (IV): R, 1-naph-
thyl, 205-206 °C; phenyl, 215-217 °C; benzyl, 179-180 °C;
phenethyl, 214-215 °C; propyl, 171-173 °C; ethyl, 179-181 °C;
methyl, 177-179 °C. The exact mass of parent ions or the
fragmentation patterns of compounds in mass spectra and
signals in NMR spectra of the synthetic compounds are consis-
tent with the structure assignments of these compounds. UV
absorptions and molar extinction coefficients of the key com-
pounds for metabolite analysis are tabulated in Table 1.
Tr ea tm en ts of An im a ls a n d P r ep a r a tion of Micr osom es.
Male F344 rats (300-400 g) from Charles River (Wilmington,
Deter m in a tion of th e F or m a tion of Th iou r ea Sid e
P r od u cts (IV) a n d Th eir Meta bolic Con ver sion s to Ur ea s
(III). In order to estimate the possible contribution of urea (III)
generated from the side product (IV) to the urea (III) obtained
in the metabolism of isothiocyanates, 2-AF was incubated with
isothiocyanates without microsomes and NADPH. At the end
of incubations, the mixtures were chilled and centrifuged. The
products (IV) were analyzed by HPLC from the supernatants
and the precipitates. Synthetic thioureas (IV) in the same
amount as the sum of the above analysis were incubated in the
same manner as for isothiocyanates metabolism and the urea
products (III) quantitated by HPLC.
In h ibition Stu d ies. In order to prove that these metabolic
conversions were mediated by P450, the CO inhibitions were
carried out as described previously (29). To prove that flavin-
containing monooxygenase was not involved in these reactions,
microsomes were heated at 40 °C for 30 min prior to the
metabolic incubations.
HP LC An a lysis of P r od u cts. Identifications of metabolite-
derived mixed ureas RNHCONHF (III) and isothiocyanate-
derived side products RNHCSNHF (IV) were based on their UV

1074 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Lee
Ta ble 1. UV Absor p tion s a n d Mola r Extin ction Coefficien ts (E) of th e Key Com p ou n d s
RNHCONHF (III) RNHCSNHF (IV)
compound, R
1-naphthyl
solvent
λmax (nm)
ꢀ
λmax (nm)
ꢀ
methanol
288
308
31 340
31 310
284
294
304
282
302
25 480
25 950
25 400
30 580
28 410
phenyl
benzyl
0.5% dimethylformamide in acetonitrile
methanol
280 (sh)
290
318 (sh)
278 (sh)
286
36 000
40 940
17 240
29 590
31 610
11 090
264 (sh)
282
292
302
264 (sh)
282
290
302
262
282
292
302
264 (sh)
282
290
302
260 (sh)
280
292
302
19 580
22 350
22 730
22 440
19 990
24 100
24 630
23 850
19 580
24 120
24 690
23 730
18 880
23 340
23 940
23 020
18 700
24 650
25 520
24 490
318 (sh)
phenethyl
propyl
1% dimethylformamide in acetonitrile
1% dimethylformamide in acetonitrile
1% dimethylformamide in acetonitrile
1% dimethylformamide in acetonitrile
288
320
30 300
10 700
286
320 (sh)
32 690
12 240
ethyl
286
318 (sh)
33 340
13 180
methyl
286
320
29 070
9 510
Ta ble 2. HP LC Reten tion Tim es (m in ) of Com p ou n d s Used in th e An a lysis of Meta bolic Con ver sion P r od u cts
solvent
RNHCONHR
RNHCSNHR
RNHCONHF
RNHCSNHF
R
systemsa
RNH2
2-AF
RNdCdS
(I)
(II)
(III)
(IV)
2-naphthyl
1-naphthyl
phenyl
benzyl
phenethyl
propyl
A
B
C
D
E
F
G
H
9.8
11.7
6.6
11.7
13.7
18.6
15.8
15.3
23.9
27.2
40.2
24.1
27.4
27.2
19.6
19.7
21.8
14.7
13.2
19.9
17.3
12.0
12.0
13.7
5.7
16.4
15.0
11.0
15.6
15.3
9.1
29.1
26.1
37.4
21.9
19.7
30.6
24.9
26.7
22.0
20.5
33.1
26.8
27.6
42.6
34.4
33.6
ethyl
methyl
∼3.5
4.7
4.3
3.3
a
Solvent systems are as described in Materials and Methods.
characteristics and retention times in HPLC profiles (Tables 1
and 2 ). Quantitation of these products was carried out by using
the external standard calibration curves constructed from the
synthetic standard compounds for each substrate. Solvent
systems used for HPLC analysis of reaction mixture from each
substrate are as follows: System A for 2-NITC: 30% methanol/
water to 68% methanol linearly in 5 min, at 68% for 27 min, to
100% linearly in 3 min; the flow rate was 1.3 mL/min and
detection was at 254 or 286 nm. System B for 1-NITC: 30%
methanol/water to 70% methanol linearly in 5 min, at 70% for
25 min, to 100% linearly in 3 min; the flow rate was 1 mL/min
and detection was at 284 nm. System C for PITC: 40%
acetonitrile/water to 44% acetonitrile linearly in 10 min, at 44%
for 30 min, to 100% linearly in 6 min; the flow rate was 1 mL/
min and detection was at 290 nm. System D for BITC: 30%
acetonitrile/water to 51% acetonitrile linearly in 5 min, at 51%
for 20 min, to 100% linearly in 5 min; the flow rate was 1 mL/
min and the detection was at 286 nm. System E for PEITC:
30% acetonitrile/water to 53% acetonitrile linearly in 5 min, at
53% for 25 min, to 100% linearly in 5 min; the flow rate was 1
mL/min and detection was at 288 nm. System F for PrITC: 30%
acetonitrile/water to 40% acetonitrile linearly in 10 min, at 40%
for 25 min, to 100% linearly in 6 min, at 100% for 3 min; the
flow rate was 1 mL/min and detection was at 286 nm. System
G for EITC: 30% acetonitrile/water to 37% acetonitrile linearly
in 5 min, at 37% for 25 min, to 100% linearly in 7 min; the flow
rate was 1 mL/min and detection was at 286 nm. System H for
MITC: 10% acetonitrile/water to 34% acetonitrile linearly in 5
min, at 34% for 30 min, to 100% linearly in 7 min, at 100% for
2 min; the flow rate was 1 mL/min and detection was at 286
nm. Data were analyzed statistically by Student’s t-test to
evaluate differences between the untreated group and treated
groups in the comparative enzyme induction studies. Prob-
ability values e0.05 were considered significant.
Resu lts
Qu a n tita tion of P r od u cts fr om Meta bolism of
2-NITC, 1-NITC, a n d BITC by Liver Micr osom es
fr om Un tr ea ted a n d In d u cer -Tr ea ted Ra ts F ed
Sem isyn th etic AIN 76A Diet w ith ou t Eth oxyqu in
or
a Con ven tion a l Gr a in Diet. For the sake of
comparison, the published data from 2- and 1-NITC and
BITC metabolized by microsomes from rats fed conven-
tional grain diet (30) are shown as part of Table 3.
1-NITC and 2-NITC were metabolized to comparable
extent and the enzymes responsible for their metabolism
were Aroclor-inducible; the metabolism of BITC was
much lower than that of the other two compounds, and
the microsomes from Aroclor-treated rats did not increase
the production of BFU (30). Further studies now showed
that although the side products, IV in Scheme 1, did form
from chemical reactions of 1-NITC or BITC with 2-AF
during the 30 min incubations, their possible maximal
contributions to the mixed urea products (1-NFU and
BFU) were estimated to be <5% when 0.2 µmol of the
mixed thioureas (IV) (possible maximal amount of side
product generated in the metabolic incubation mixtures)
were incubated with NADPH and microsomes in the
similar manner.
Comparative induction studies were carried out with
4 inducers using rats fed semisynthetic diet AIN 76A

Oxidative Desulfuration of Isothiocyanates
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1075
Ta ble 3. Meta bolic F or m a tion of Isocya n a tes fr om Isoth iocya n a tes in th e P r esen ce of 2-AF by In d u ced or Un in d u ced
Liver Micr osom es fr om Ra ts F ed Con ven tion a l Gr a in Diet or AIN 76A Diet w ith ou t Eth oxyqu in a
product (nmol/0.4 mg of protein/30 min)
source of microsomes:
inducer (no. of rats)
diet
incubation conditions
2-NFU
<0.1
<0.1
1.19 ( 0.29
0.38 ( 0.32
11.60 ( 2.73
0.07 ( 0.12
9.07 ( 0.83d
0.26 ( 0.28
3.36 ( 0.14d
0.21 ( 0.20
2.65 ( 0.70e
0.31 ( 0.36
3.57 ( 0.58d
0.34 ( 0.41
1-NFU
BFU
none (2)
conventional grainb
complete system
-NADPH
1.17, 1.15
1.12, 0.38
2.10, 2.14
0.65, 0.61
none (4)
AIN 76Ac
complete system
-NADPH
1.11 ( 0.10
0.26 ( 0.08
11.65 ( 1.65
0.27 ( 0.22
11.12 ( 1.15f
0.36 ( 0.19
5.50 ( 0.85e
0.27 ( 0.05
1.88 ( 0.30g
0.22 ( 0.05
2.78 ( 0.07d
0.24 ( 0.07
1.39 ( 0.10
0.32 ( 0.15
1.95 ( 0.17
0.35 ( 0.08
2.30 ( 0.35g
0.26 ( 0.13
1.17 ( 0.14
0.29 ( 0.14
1.58 ( 0.22
0.31 ( 0.02
1.85 ( 0.17f
0.32 ( 0.05
Aroclor (3)
conventional grainb
AIN 76Ac
complete system
-NADPH
Aroclor (3)
complete system
-NADPH
â-naphthoflavone (3)
isosafrole (3)
phenobarbital (3)
AIN 76Ac
complete system
-NADPH
AIN 76Ac
complete system
-NADPH
AIN 76Ac
complete system
-NADPH
a
Complete system consisted of 0.5 µmol of substrate, 0.4 mg of microsomal protein, 5 µmol of NADPH, and 0.2 µmol of 2-AF in 1 mL
b
of solution. 2-NFU, 1-NFU, and BFU are products from metabolic conversion of 2-NITC, 1-NITC, and BITC, respectively. Incubation
mixtures of each substrate with microsomes from rats fed the conventional grain diet were analyzed by HPLC. Data are mean ( standard
deviation obtained from microsomes from 3 rats or the numbers from microsomes from 2 rats (30). c Incubation mixtures of each substrate
with microsomes from rats fed AIN 76A without ethoxyquin were analyzed by HPLC using systems A, B, and D, respectively, as described
d
in Materials and Methods. Data are mean ( standard deviation obtained from microsomes from 3 rats. The values are significantly
higher than the uninduced control values, since the p values of the Student’s t-test on the values of the complete system are <0.0008.e p
g
<0.013. f p <0.006. p e0.05.
without ethoxyquin. As shown in Table 3, all four
inducing agents significantly increased the formation of
2-NFU from metabolism of 2-NITC in the order of Aroclor
. phenobarbital g â-naphthoflavone > isosafrole under
the present protocol. The formation of 1-NFU from the
metabolism of 1-NITC was also increased by the four
inducers in the order of Aroclor . â-naphthoflavone >
phenobarbital > isosafrole. Aroclor increased the pro-
duction of BFU from the metabolism of BITC ∼2-fold,
and only a slight increase was observed with the pheno-
barbital-induced microsomes. Treatment of these rats
with â-naphthoflavone or isosafrole did not increase the
F igu r e 1. Time course of metabolic conversion of 1-naphthyl
production of BFU. Consistent with experiments with
2-NITC (29), BFU formation from the metabolism of
BITC was inhibited by CO (CO:O2 ) 80:20) by ∼40% as
compared to the experiments carried out in air when
Aroclor-induced microsomes were used (data not shown).
Qu an titation of P r odu cts fr om Com par ative Meta-
bolic Con ver sion of Isoth iocya n a tes to Isocya n a tes.
Since Aroclor was the best inducer so far tested under
the current induction protocol and the metabolic conver-
sion assay procedure, the pooled microsomes from 6
Aroclor-treated rats fed conventional grain diet were used
to study the metabolic potentials of 1-NITC, PITC, BITC,
PEITC, PrITC, EITC, and MITC. The time course was
from 0 up to 30 or 60 min using the procedure described
in Materials and Methods. Figure 1 shows the results
from 1-NITC as an example. The linear velocity in the
complete reaction reached maximum at 5 min in all cases.
The controls without NADPH showed very little or no
activity. Table 4 shows the comparative metabolic
conversion of these 7 compounds based on the initial
linear rates (5 min) of the formation of the ureas (III) as
a measure of the extent of isocyanate metabolite forma-
tion. Metabolic potentials followed the order of 1-NITC
> PITC > BITC, PEIT > PrITC, EITC, and MITC. In
the case of MITC, the HPLC profile was more complicated
and the urea (MFU) formation was so little that the
result from only one good HPLC run was shown in Table
4.
isothiocyanate. Metabolic conversion of 1-NITC to 1-naphthyl
isocyanate; trapping by 2-AF to form 1-NFU and HPLC analysis
of 1-NFU was as described in Materials and Methods.
Ta ble 4. Com p a r a tive Rea ctivities of Meta bolic
Con ver sion of Isoth iocya n a tes to Isocya n a tesa
nmol of product/
(mg of protein‚min)
2-AF-derived
substrates
(0.5 mM)
urea product
RNHCONHF (III)
complete
reaction
-NADPH
control
1-NITC
PITC
BITC
PEITC
PrITC
EITC
1-NFU
PFU
BFU
PEFU
PrFU
EFU
2.27 ( 0.06b
1.26 ( 0.20
0.82 ( 0.06
0.73 ( 0.02
0.07 ( 0.03
0.13d
0.15 ( 0.02
∼0.12c
0.04 ( 0.005
0.03 ( 0.005
∼0.01c
0 ( 0
MITC
MFU
0.03e
0e
a
The reactivities are based on initial linear reaction rates at 5
min for the conversion of isothiocyanates to isocyanates with liver
microsomes from Aroclor-treated rats. The metabolites were
trapped by formation of ureas with 2-aminofluorene (0.2 mM).
b
Data are from HPLC analysis of the 2-AF-derived ureas at 5 min
using solvent systems as described in Materials and Methods.
Standard deviations are shown for data from 3 or more samples.
c This is taken from the -NADPH control time course curve at 5
d
min. This is the average result from 2 samples. e This is the
result from 1 sample.
cya n a tes to Isocya n a tes. Except for the 3 aliphatic
isothiocyanates, at the end of 5 min incubations, some
side product (IV) did form when NADPH and microsomes
were omitted from the complete metabolic incubation
mixtures (∼105 nmol from 1-NITC, ∼15 nmol from PITC,
BITC, and PEITC, and none from PrITC, EITC, and
P ossible Con tr ibu tion of Ur ea s (III) d u e to th e
F or m a tion of th e Sid e P r od u ct Th iou r ea s (IV) in
th e Com p a r a tive Meta bolic Con ver sion of Isoth io-

1076 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Lee
Ta ble 5. Effects of CO a n d Hea tin g of Micr osom es on th e
P r od u ction of RNHCONHF (III) fr om Meta bolism of
Isoth iocya n a tesa
Results of BITC metabolism by microsomes from rats
fed AIN 76A diet without antioxidant show that, indeed,
formation of BFU from the uninduced microsomes de-
creased as compared to that from rats fed conventional
grain diet and the use of the Aroclor-induced microsomes
now gave approximately twice the amount of BFU as
compared to that of the uninduced control. Phenobar-
bital-induced microsomes also slightly increased the
formation of BFU, suggesting that P450 2B1 and 2B2
may be involved in the metabolism of BITC. This is
consistent with the finding that the microsomes from the
untreated rat fed the conventional grain diet gave the
same amount of BFU as that treated with Aroclor (see
Table 3), since ethoxyquin in the diet served as inducing
agent for P450 2B1 and 2B2 (31) and probably P450 3A2
induced by the same agent may also catalyze the meta-
bolic conversion of BITC. These data suggest that
Aroclor was the only effective inducer for the metabolism
of BITC and feeding animals with semisynthetic diets
may be required in future studies where the identification
of inducing agents capable of increasing the metabolism
of isothiocyanates is sought. Results from this study
have established that metabolism of BITC is mediated
by P450(s) and not by the flavin-containing monooxy-
genase.
RNHCONHF (%)
isothio-
unheated
heated
cyanate N2:O2 ) 9:1 CO:O2 ) 9:1 microsomes microsomes
1-NITC
PITC
BITC
PEITC
EITC
100
100
100
100
100
40 ( 2
39 ( 4
47 ( 3
29 ( 11
51 ( 2
100
100
100
100
100
96 ( 5
95 ( 3
100 ( 12
112 ( 8
106, 91
a
Experiments were carried out using liver microsomes from
Aroclor-treated rats as described in Materials and Methods.
Results of CO inhibition in the atmosphere of CO:O2 ) 9:1 were
compared with that from the controls in N2:O2 ) 9:1. Results from
heated microsomes were compared with those from unheated
microsomes as controls. Data are from triplicates (with standard
deviations) or from duplicates.
MITC). These quantities of synthetic thioureas from
these four aryl and arylalkyl isothiocyanates were enzy-
matically incubated in the same manner as for the
isothiocyanates and the urea (III) products analyzed.
These results were compared with the amounts of urea
(III) obtained from enzymatic incubation of isothio-
cyanates in each case, and their contributions are no
more than 4.6-7.2%.
In h ibition Stu d ies. As shown in Table 5, the meta-
bolic conversion of these isothiocyanates (PrITC and
MITC were not studied due to their low metabolic
potentials) was inhibited by CO, and heating of mi-
crosomes did not diminish the product formation.
â-Naphthoflavone, isosafrole, and phenobarbital are
known to induce primarily P450 1A1, P450 1A2, and
P450 2B1 and 2B2, respectively (38). As shown in Table
3, the formation of 1-NFU and 2-NFU from Aroclor-
induced microsomes may involve all of these P450s, since
each of these agents increased the metabolism of 1-NITC
and 2-NITC. P450 1A1 may be more important for
metabolism of 1-NITC than for 2-NITC, since â-naphtho-
flavone-induced microsomes gave higher amount of 1-NFU
than 2-NFU.
Discu ssion
Aroclor 1254 is known to induce substantial amounts
of P450 1A1, 1A2, 2A1, 2B1, and 2B2 (38). In the initial
study of the oxidative desulfuration of 2-NITC to 2-naph-
thyl isocyanate (29), liver microsomes used were prepared
from rats fed a conventional grain diet before and during
the Aroclor induction period. There was an increase in
the production of 2-NFU of ∼10-fold with the induced
microsomes. Extension of this metabolic conversion
study to 1-NITC and BITC using microsomes from rats
fed conventional grain diet (30; part of Table 3) suggested
that Aroclor-induced enzymes increased metabolism of
both 1-NITC and 2-NITC but was without effect on BITC.
It was reasoned that some component(s) of the grain diet
might have acted as an inducer(s) in the untreated
animals, and since the metabolism of BITC was low, that
any additional induction effect due to Aroclor was not
manifested in the treated animals. Alternatively, an-
other enzyme system, such as flavin-containing mono-
oxygenase, might be responsible for the metabolism of
BITC. The purpose of the present comparative induction
study was threefold: The first was to find out if the
enzyme(s) for metabolic conversion of BITC was induc-
ible; the second was to gain insight into which enzyme(s)
is most likely responsible for the metabolic conversion
of these compounds; the third was to find a good inducer
to screen for the metabolic potentials of more isothio-
cyanates. To minimize the possible induction effects on
the control animals from the conventional grain diet, the
animals were fed semisynthetic AIN 76A diet that had
been prepared without ethoxyquin, an agent which
induces P450 2B1, 2B2, 1A2, and 3A2 (31), that is usually
added to serve as an antioxidant to protect the corn oil
of the diet.
The experimental approach of trapping the reactive
isocyanate metabolites with 2-AF is a complex chemical
system and, although useful, is not perfect, and cannot
serve as a measure of the absolute activity of the
microsomal proteins toward these isothiocyanate sub-
strates since there are factors that may affect the
estimation of the total amount of the isocyanate metabo-
lites formed. First, hydrolysis of isocyanates leads to the
formation of symmetrically disubstituted ureas (I) and
thioureas (II) in the cases of 2-NITC and 1-NITC, which
occurred even in the presence of 2-AF. However, in the
absence of 2-AF, neither dibenzylurea nor dibenzylthio-
urea was detected in the conversion of BITC to its
isocyanate. This is due to their low molar extinction
coefficients (this applies to all arylalkyl and aliphatic
isothiocyanates). Therefore, for comparison purposes,
this trapping method was adopted in order to be able to
detect benzyl isocyanate formed in the metabolism of
BITC as BFU. Second, 2-AF is also known to be the
substrate for rat liver microsomes and purified P450s
from rats treated with â-naphthoflavone, isosafrole, and
phenobarbital (39). Hence, the trapping agent, 2-AF (0.2
mM, with the maximal trapping capability in the ranges
of 0.05-0.6 mM of 2-AF tested in 1-NITC metabolism
using Aroclor-induced microsomes), may compete for
P450 with the isothiocyanate substrates. Since isos-
afrole-induced P450 (1A2) had the highest activity in the
metabolism of 2-AF (38), the activity toward the isothio-
cyanates may be more affected in this case. Another
factor that may reduce the activity of isosafrole-induced
microsomes is the formation of a P450 complex with the
metabolites of isosafrole causing the partial loss of the

Oxidative Desulfuration of Isothiocyanates
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1077
enzyme activity (40). Removal of the metabolites of
isosafrole from possible P450 complexes was not at-
tempted in the present study. Furthermore, 2- and
1-aminonaphthalenes may compete for enzymes once
they are formed in the incubation mixtures of 2- and
1-NITC through hydrolysis of isocyanate metabolites,
since these amines are also substrates for P450s induced
by these agents, particularly, â-naphthoflavone and
isosafrole (39).
years, the mechanism of the inhibition of chemical
carcinogenesis by isothiocyanates has been extensively
studied with nitrosamines (reviewed in refs 44-46). The
inhibitions of tumorigenicity and DNA adduct formation
of these procarcinogens are due to the chemical inactiva-
tion and competitive inhibition of the reqired P450
enzymes. This is consistent with the fact that isothio-
cyanates can react with protein and may be metabolized
by P450 enzymes as described in this report, thus,
competing for the activation enzymes required by the
carcinogens. However, the possible genotoxic conse-
quences of the activation of isothiocyanates by P450’s
remain largely unexplored.
Results from the comparative metabolic conversion
studies using microsomes from Aroclor-treated rats fed
conventional grain diet (Table 4) reveal that the highly
aromatic 1-NITC had the highest metabolic potential
followed by the monocyclic aromatic PITC, the metabolic
activities were futher diminished for BITC and PEITC,
and aliphatic PrITC, EITC, and MITC were only very
weakly metabolized. However, the low reactivity of the
aliphatic isothiocyanates may be partly due to the
relatively volatile nature of their isocyanate metabolites,
possible decomposition in the incubations, and more
importantly, the major enzymes required for the meta-
bolic conversion of these compounds may not be present
in the Aroclor-induced microsomes used in these experi-
ments. Metabolic potentials of isothiocyanates will have
to be explored further with other families of P450
enzymes. Experiments using reconstituted specific P450
enzymes and/or specific antibodies against P450 enzymes
are required to identify the specific enzymes involved in
the metabolism of these compounds.
Ack n ow led gm en t. The author wishes to thank Mr.
K. Zukowski for assistance in preparations of some
microsomes and Ms. E. Thomas-Weber for assistance in
the preparation of the manuscript. The author would
also like to thank Dr. M. B. Ksebati and Ms. M. B. Kempff
from the Central Instrumentation Facility of the Com-
prehensive Cancer Center of Metropolitan Detroit for
measuring the NMR and mass spectra, respectively.
This contribution from the A. Alfred Taubman Facility
was supported by American Cancer Society Grant CN-
78 and an institutional grant from the United Way of
Detroit.
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Unpublished observations.

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