0090-9556/86/1401-0097$02.00/0 DRtuI
METABOLIsM
AND
Vol. 14, No. 1 in USA.
DISPOSITION
© 1986 by The American
Copyright
FORMATION
Society
OF
for Pharmacology
and Experimental
METHYLTHIO
Therapeutics
METABOLITES
OF
THE M. J. BARTELS, Institute
Research,
July 1 8, 1 985;
INDENE
IN THE
GUINEA
PIG
AND
RAT
E. C. HORNING,
for Lipid
(Received
Printed
AND
Baylor
accepted
M.
College
G. HORNING
of Medicine
September
23, 1985)
ABSTRACT: The formation
of methylthio
to be a significant
metabolites
route
of this metabolic
aspects
isomers
of epoxides
of metabolism
conversion
in some
for indene
of hydroxy(methylthio)indane
the administered
dose
shown
were examined.
were
found
were synthesized and administered to the guinea pig. The methylthio metabolite I was present as a significant urinary metabolite of both
Several
in the
Two
urine
conjugates
of
(14.3 and 100 mg/kg, ip). The major (I) was present as 6-9% of
after 24 hr, while
lower
amounts
(0-0.6%)
and
mercapturic
acid
(I-MER)
conjugates
of indene
have
dihydrodiol
also
been
intermediate lites.
in the
Reactive
thiazole
(5),
and
bromobenzene
suggesting
formation
compounds
from
and
styrene
the formation
aromatic
indene.
amounts of2-hydroxy-l-methylthioindane(isomer from indene, as well as its reactive metabolite the guinea
pig.
metabolism tion
ofthe
anistic
served aspects
been
ofthe
metabSignificant
I) were found indene oxide, in
glutathione
pathway
previously oxide
also
by in vitro
in the
identifica(8) as well
acids as urinary metabolites of rats (9). We therefore examined the further of the glutathione pathway to methyl-
of indene
and
as precursors of this
shown
conjugate
of mercapturic the epoxide of conjugates
metabolites
MER
of the
has
glutathione
as isolation administered conversion thio
Involvement
of indene
found
that
for the formation
pathway
both
I-GLU’
of isomer
and
I-
I. Mech-
are discussed.
This work was supported
by Grant 0-125 of the Robert A. Welch Foundation Institute of General Medical Sciences. I Abbreviations used are: 1-GLU and I-MER, glutathione and mercapturic acid conjugates, respectively, of indene oxide; BSTFA, bistrimethylsilyltnfluoroacetamide; GLC, gas-liquid chromatography; TMS-tnmethylsilyl-; MU, methylene unit; El-MS. electron impact mass spectrometry; FAB-MS, fast atom bornbardrnent mass spectrornetry. and by Grant GM 13901 of the National
Send Laboratory,
reprint
1803
requests Building,
to: Mkhael Dow
Chern
Bartels, Ph.D., Toxicology Co., Midland, Ml 48674.
J.
of the dose These
and indene via further
results
is a signifi-
oxide
in the guinea
metabolism
of conju-
and Methods
Reagents and other chemicals were obtained from the following sources: indene, Eastman-Kodak Co. (Rochester, NY); methanethiol (1.5 M in methanol), l-phenylethane-l,2-diol, sodium methoxide, and mchloroperbenzoic acid, Aldrich Chemical Co. (Milwaukee, WI); N-acetylL-cysteifle, N-acetyl-i-methionine, glutathione, and fJ-glucuronidase (type H-2, I 1 1,000 units l-glucuronidase and 6300 units sulfatase/ml), Sigma Chemical Co. (St. Louis, MO); and silylating reagents, Pierce Chemical Co. (Rockford, IL). All solvents and other reagents were reagent grade or chromatographic grade. Male Sprague-Dawley rats were obtamed from Timco Breeding Labs (Houston, TX) and male Hartley guinea pigs were obtained from CAMM Research Institute (Wayne, NJ). Administration of Compounds to Guinea Pigs. Male Hartley guinea pigs (625-800 g) were administered indene (14.3 and 100 mg/kg) and indene oxide ( I 2.5 and 100 mg/kg) in 0.5 ml ofcorn oil, ip. 1-GLU ( 1 1.6 and 18.5 mg/kg) and I-MER (8. 1 mg/kg) were administered to male Hartley guinea pigs in 0.5 ml of distilled water, ip. Following injection, the guinea pigs were housed individually in metabolism cages, with water only, and 0-24 hr urine samples were collected. The pH of the urine samples was adjusted to 4.5-5.0 (acetic acid and 200 mg sodium acetate) before storing at -20’C. Administration of Indene Oxide to Rats. Male Sprague-Dawley rats (200-210 g) were given indene oxide (100 mg/kg) in 0.5 ml of corn oil by ip injection. The rats were housed individually in metabolism cages, with water only, and 0-24 hr urine samples collected. The pH of the urine samples was adjusted to 4.5-5.0 (acetic acid and 20 mg sodium acetate) before storing at -20’C. Isolation of Metabolites from Urine. Aliquots of urine samples (1 ml) from the guinea pig or rat were incubated with 50 zl of fJ-glucuronidase, 18 hr at 37#{176}C, to hydrolyze conjugates ofthe metabolites. The hydrolyzed urine samples were spiked with 20 zg of l-phenylethane-l,2-diol (1 mg/ ml in methanol) as an internal reference compound, saturated with ammonium carbonate, and extracted with 5 ml of ethyl acetate. The solvent was evaporated (N2), and the residue was dissolved in 30 l of pyridine and silylated with 30 l of BSTFA at 60’C for 1 hr. Aliquots of the derivatized samples were then analyzed by GLC and GC-MS.
is an
(7)
of methylthio
hydrocarbon
arises
Materials
metabo-
oxide
and 5.7%
gates in the glutathione pathway. In the rat, isomer I is a minor metabolite. Mechanistic aspects of the formation of these thioether metabolites are discussed.
(4)
oxide
methylthio
9.6%
oxide
dihydrodiol
an arene
for indene
pig, and that this metabolite
as 2-acetamido-4-chloromethyl(6),
metabolites. we examined the
that
of aromatic such
1,3-dichloropropene
form methylthio In this study olites
(3)
identified,
comprising
of a hydroxy(methylthio)indane
cant route of metabolism
of
The formation of methylthio metabolites has been found to be a significant route of metabolism in some species for many xenobiotics. Aromatic compounds such as bromazepam ( 1), tetrachlorobiphenyl (2), and naphthalene (3) are converted to methylthio metabolites in vivo. The methylthio analogs of naphthalene
oxide,
show that the formation
a minor isomer (II) were observed. A significant amount of isomer I was found as a urinary metabolite of indene oxide (14% of 12.5 mg/ kg, ip). To further elucidate the route of formation of I, the glutathione (1-GLU)
of indene
of 1-GLU (5 mg, ip) and 1-MER (4 mg, ip), respectively.
Research
Gas
Chromatography
were carried 97
out with
and GC-MS. a 47-m
SE-30
open
Gas chromatographic tubular
glass
capillary
analyses column
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2015
guinea pigs administered indene isomer, 2-hydroxy-1-methyfthioindane
has been species.
BARTELS
98
B varied
linearly
from
10 to 90%.
The
programming
time
for the
separations was 20 mm with a flow rate of 1.5 mI/mm. Nuclear Magnetic Resonance Spectrometry. Both ‘H- and ‘3C-NMR spectra were obtained with an NMC-1280 300-MHz instrument (Nicolet Magnetics Corp., Fremont, CA), with tetramethylsilane as an internal standard.
AL.
resulting
to
15
was heated at 60#{176}C for 2 hr. evaporated (N2), diluted and extracted with ether (four times at 10 ml). The
solution ml (H20),
combined ether extracts were evaporated (N2) and purified by semipreparative reverse phase HPLC to yield 93.8 mg (45%) of 2-hydroxy-lmethylthioindane as a clear oil; ‘3C NMR (CDCI3) 6 140-125 (6, Art), 77.9 (l,QHOH), 57.4 (1, HSCH3), 39.7 (1, #{231}H2),12.9 (1, SH3); ‘H NMR (CDCI3) 5 7.37-7.09 (m, 4, Arj.J), 4.46 (m, 1, CFJOH), 4.03 (d, I, CUSCH3, J = 4.4 Hz), 3.29 (dd, 1, CIJH, J = 6.3 and 16.4 Hz), 2.84 (dd, 1 , CH1J, J = 5.2 and 16.4 Hz), 2.05 (s, 3, SCIJ3). A portion (1 mg) ofthe product was dissolved in 30 l ofpyridine and derivatized with 30 il of BSTFA at 60#{176}C for 1 hr. Capillary GLC of the TMS-derivative (MU = 16.29) showed the product to be greater than 99% pure; El-MS (m/:) 252 (3%, M), 237 (18%), 205 (81%), 189 (20%), 162(51%), 147(12%), 115 (basepeak). Synthesis of S-(Hydroxyindanyl)glutathione. Indene oxide (331 mg, 2.5 mmol), in 3 ml of dimethyl sulfoxide, was added to a solution containing 615 mg (2 mmol) glutathione in 25 ml ofwater(pH 7.4). The resulting solution was stirred under nitrogen at 25C for 48 hr, washed
ethyl acetate (five times at 20 ml), concentrated to 3 ml, and adjusted to pH 3.5 (2 M Ha). The resulting oil was applied to a column (1 1 x 300 mm) containing 150 g of XAD-2 resin and eluted with water with
(600
ml),
methanol:water,
1: 1 (v/v)
(600
ml),
and
methanol
(600
ml).
The fractions containing product, by TLC (1 1), were combined and evaporated to yield an oil. This product was triturated with acetone and crystallized from wateracetone to yield 160 mg (18%) of S-(hydroxyindanyl)glutathione, 422
(0.3%),
m.p. 308
(1%),
(m/z)
18 1-1 82#{176}C; FAB-MS 167
(4%),
147
(4%),
133
(8%),
440
(4%,
MH),
1 15 (5%).
Synthesis of S-(Hydroxyindanyl)mercapturic acid. Indene oxide (662 mg, 5 mmol), in 4 ml of dimethyl sulfoxide, was added to a solution containing 653 mg (4 mmol) N-acetyl-L-cysteine in 50 ml of water (pH 7.4). An additional 10 ml of dimethyl sulfoxide was added and the resulting solution was stirred under nitrogen at 25#{176}C for 20 hr. The reaction mixture was washed with ethyl acetate (five times at 100 ml),
concentrated
to 20 ml, and adjusted to pH 3.5. The resulting oil was (36 x 300 mm) containing 200 g of XAD-2 resin and eluted with water (600 ml), methanol:water, 1:1 (v/v) (600 ml), and methanol (700 ml). The fractions containing product, by TLC (1 1), were applied
to a column
Synthesis of Indene Oxide. Indene (5.6 g, 48.2 mmol), in 30 ml of dichloromethane, was added dropwise to a stirred solution of 10.4 g(60.3 combined and evaporated to yield a gum. This material was further mmol) m-chloroperbenzoic acid in 150 ml of dichloromethane at 0#{176}C. purified by semipreparative reverse phase HPLC to yield 16 mg (1.4%) The resulting solution was stirred at 0#{176}C for 2 hr. warmed to room of S-(hydroxyindanyl)mercapturic acid; FAB-MS (ml:) 296 (87%, temperature and filtered. The filtrate was washed with 5% aqueous MH),278(basepeak), 164(30%), 133(51%), 115(11%). Na2SO3 (twice at 50 ml), 5% aqueous Na2CO3 (twice at 50 ml), water Synthesis and Decomposition of S-(Hydroxyindanyl)-N-acetyl-L-me(twice at 50 ml), dried (K2CO3). and evaporated to yield a yellow oil. thionine. S-(Hydroxyindanyl)-N-acetyl-L-methionine was prepared with Vacuum distillation afforded 2.5 g (39%) of indene oxide, b.p. 37#{176}C the procedure developed for the analogous styrene conjugate (12). The (0.025 torr): El-MS (m/:) 132 (18%, M’), 104 (base peak), 78 (86%), 77 reaction product was purified by semipreparative reverse phase HPLC. (65%), 63 (23%). 5 1 (57%). Capillary GLC showed no additional prodAn FAB mass spectrum of the mixture of isomers obtained showed an ion at ml: 324, which corresponds to the protonated sulfonium comucts. Synthesis of cis- and trans-Indane-l,2-diol (III and IV). Indene oxide pound(fig. 1). A portion of the sulfonium product (50%) was dissolved in 0.5 ml of (200 mg, 1.52 mmol) was added to a solution containing 2 ml of0.5 M acetone and heated (open tube) in a boiling water bath for 4 mm. The aqueous sulfuric acid and 2 ml oftetrahydrofuran in a 5-ml thick-walled glass vial. The vial was sealed and the solution was heated at 50#{176}C for 20 residue was dissolved in 100 l ofpyridine and derivatized with 50 zl of hr. The resulting mixture was diluted to 10 ml (H2O), adjusted to pH 10 BSTFA at 60#{176}C for 1 hr. Capillary GLC showed the major product to be (NH4OH), and extracted with ether(twice at 20 ml). The combined ether 2-hydroxy-l-methylthioindane(I), which indicates that the major sulfonium adduct was S-(2’-hydroxy-l-indanyl)-N-acetyl-L-methionine. Both extracts were dried (MgSO4) and evaporated to yield a crystalline solid. Crystallization from benzene yielded 48 mg (21%) of trans-indane-l,2isomers of indane-l,2-diol (cis-diol, III; irans-diol, IV) were identified in smallamountsas theirTMS-derivatives. A fourth decomposition product diol (IV) as off-white needles, m.p. 153-154#{176}C[literature (10) m.p. 158was observed (MU 16.47) which had a mass spectrum very similar to l59#{176}Cl. A portion (I mg) ofthe product was dissolved in 30 zI of pyridine and derivatized with 30 I of BSTFA at 60#{176}C for 1 hr. Capillary GLC of metabolite I. This compound was also observed as a metabolite of indene in the guinea pig and was tentatively assigned the structure of l-hydroxythe TMS-derivative (MU = 16. 15) showed the product to be greater than 2-methylthioindane (II); El-MS of the TMS-derivative (m/z) 252 (1 1%, 97% pure; El-MS (m/:) 294 (19%, M), 279 (21%), 203 (41%), 147 (base peak), 131 (13%), 1 15 (51%). A minor product (MU = 15.83) M), 237 (32%), 205 (95%), 189 (24%), 162 (base peak), 147 (16%), 115 present in the sample was identified by GC-MS as cis-indane-l,2-diol (84%). Relative yields of compounds I-IV were 100:6:3:1, respectively. A second portion ofsulfonium product (50%) was dissolved in 0.5 ml (III): El-MS (m/:) 294 (19%, M), 279 (19%), 203 (29%), 147 (base peak). 131 (I 1%), I 15 (51%). of water, decomposed in a boiling water bath, and derivatized as above. Synthesis
of 2-Hydroxy-I-methylthioindane
(I). Indene
oxide
(155
mg. I . 17 mmol), in 5 ml ofmethanol, was added to a solution containing 149 mg (3. 1 mmol, 2. 1 ml ofa 1.5 M solution in methanol) methanethiol and 32.5 mg (0.6 mmol) sodium methoxide in 10 ml of methanol. The
Analysis by capillary GLC again yielded the same four compounds, with the two indane-l,2-diol isomers as major products. Relative yields of compounds
I-IV
A subsequent
were 30:4:100:12,
experiment
respectively.
was performed
in which
isomer
I was
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2015
and flame ionization detection. The flow rate for the carrier gas (helium) was 1.2 mI/mm. Temperature programming was used (l40#{176}-220#{176}C at 2’/min) and MU values were obtained with reference n-alkanes. Mass spectrometric studies were carried out with a Nermag R-lO 10-C gas chromatography-spectrometer using a 2 m x 2 mm column with 5% SE-30 packing. The flow rate for the carrier gas (helium) was 12 mI/mm. Separations were accomplished with temperature programming, 155#{176}220#{176}C at 2#{176}/mm.The instrumental parameters were: ion source temperature, 220#{176}C: ionizing current, 0.2 mamp; electron energy, 50 eV. Fast Atom Bombardment Mass Spectrometry. A modified Finnigan 101 5 mass spectrometer was employed. A Saddle Field FAB-l 1F-GG ion gun (Ion Tech Ltd., Teddinglon, England) was attached to the housing at the gas chromatographic inlet site, and an open source was constructed in our laboratory. Argon was used as the neutral gas. The probe was stainless steel; the angle of incidence of the argon beam with the probe tip was 60#{176}. The ion gun voltage was 4 kV: the limiting current ws 2 mamp, and the equivalent ion current of the neutral beam was 20 amp. All samples were dissolved in methanol and added to a glycerol matrix on the probe tip. An RDS-Nermag unit (Nermag Co., RueilMalmaison, France) was used as a data system. High Performance Liquid Chromatography. Reverse phase HPLC analyses were carried out by gradient elution with a dual solvent delivery system (Waters Associates models 6000A and M-45), a solvent programmer (Waters model 660), and a UV absorbance detector (Waters model 440) set at 254 nm. Omniscribe recorders(Houston, TX) were employed. Separations were carried out with a C18 zBondapak analytical column (30 cm X 3.9 mm) and with a semipreparative column (30 cm x 7.8 mm) (Waters) with the same type of packing. The solvent system consisted of: solvent A, methanol/water/acetic acid (20:80:0. 1), and solvent B, methanol/water/acetic acid (80:20:0. 1 ). The amount of solvent
ET
METHYLTHIO
METABOLITES
OF
INDENE
99
r’1O
104 100-
.12.6
192 H3C
R
+ -....
S
75.
1u__J
144 133 50-
.6
.!
117
1
2::]LL_____ 9,)
190
140
240
290
&-1 1
340
m/z
1 . FAB
FIG.
The
ions at in/:
mass
are due to protonated
324
spectrum
(glycerol
zwitterion
matrix)
of S-(hydroxvindanyl)-N-acetyl-L-methionine.
structures.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2015
‘I
CH,S
H4,
CH3S
H,,3
\/
4.2
4.4
4.0
3.8
3.4
3.6
3.2
3.0
28
-8
.g
4.4
4.2
4.0
3.8
3.4
3.6
3.0
3.2
2.8
PPM
7
8
FIG.
3.
Abscissa,
isomer 4.4
FIG.
4.2
2. ‘H-NMR
4.0
spectra
3.8
3.4
3.6
ofihefour
‘
3.0
of 2-hvdrox
v-I-
(I).
spectra of the C-I, C-2, and C-3 protons of spectra ofthe same protons following irradia‘H-NMR spectra ofthe same protons following
synthesized without purification of the sulfonium intermediate. Indene oxide (43.8 mg. 0.33 mmol) was added to a solution containing 100 mg (0.52 mmol) N-acetyl-i-methionine in 3 ml of acetone. The resulting solution was sealed and heated at 40#{176}C for 18 hr. The reaction mixture was evaporated (N2), dissolved in I M aqueous NaOH, and extracted rapidly with ethyl acetate (twice at 3 ml). The combined ethyl acetate
extracts Purification
were dried
(MgSO4)
by semipreparative
and
evaporated
reverse
phase
to afford HPLC
‘H-NMR I. The contour
5
6
4
3
2
PPM(’H)
proton-carbon chemical shtft correlation of2-hydro.yl-methylthioindane (I). spectra of isomer I: ordinate, plot correlates proton-carbon
‘3C-NMR
NMR
spectra
of
attachments.
2.8 PPM
ei’clopenit’lprotons
methvlthioindane Top, normal ‘H-NMR isomer I; center, ‘H-NMR tion at 4.03 ppm; bottom. irradiation at 4.46 ppm.
3.2
Heteronuclear spectrum
a brown
afforded
oil.
I I mg
(19%) of isomer I. Analysis of the showed the product to be 94% pure (6%).
TMS-derivative with isomer
by capillary GLC II as a minor product
Results The structure of the major methylthio metabolite of indene in the guinea pig was determined with GLC and (iC-MS. This compound, 2-hydroxy-l-methylthioindane (I), was identified by comparison with a synthesized reference compound. Confirmation of the structure of the synthetic product required multinuclear NMR experiments. Decoupling experiments were initially performed to confirm the assignment of the two aliphatic methine protons of isomer I (fig. 2). Irradiation of the doublet at 4.03 ppm altered only the signal at 4.46 ppm, which suggested that the signal at 4.03 ppm
100
BARTELS
ET
AL.
A
294
115 100-
205
rn/i 75 -
162
252
50.
25
I
91
189
237
II
I
.
.
,.
80
Fu;.
,
.
130
4. IsIass
spec/ruin
ofihe
ion is present
T.%IS-derivative (I).
at ,n/:
)
B
,
294
280
of2-livdro.vv-
Irn/i
252.
252
I
and diol ,n’tabolitt pig and rat
in tile guinea
itidene
0/
scan Time
Metabolites
Compound .
Dose
.
Administered
II
extract.
dose
9.0. 9.0 5.9. 6.5 12.7. 15.3 3.4. 4.4
0.3. 0.9 0.2. 0.3 0.2.0.3 0. 0
1-GLU”
11.6
6.6. 12.6
0.0
0,0
0,0
lGLUa
18.5
lI.1,I2.3
0,0
I-MEW
8.1
U
Values
100.0
are percentage
samples,
0.4. 1.4 0.9, 1.1 1.4, 1.7 1.8, 2.1
0,0
0,0
5.7±0.8
0
0
0.3 ± 0.2
0
7.3
of metabolite
present
in 0-24
2. h Values as in footnote a but n = 3. Metabolite present in 0-24 hr urine indene oxide, ii = 4. urine
0 ± S
9.8
± 2.2
hr guinea
pig
ii =
samples
of rats
administered
methine
proton
at 4.03
ppm.
The
secondary
methine
proton at 4.46 ppm is similarly linked to the hydroxyl-bearing carbon at 77.9 ppm. These results confirm the structure of isomer I as 2-hydroxy-l-methylthioindane. The sulfonium salt, S-(2’-hydroxy-l-indanyl)-N-acetyl-L-methionine
(fig.
1), along
with
a small
amount
of the
positional
isomer, was prepared as an intermediate in the synthesis of isomers I and II. The product obtained from HPLC purification was a mixture of isomers, as shown by the presence of isomers I and II as decomposition products. Decomposition ofthis sulfonium
compound,
in both
organic
and
aqueous
solutions,
250
381
360
8:28
9:34
10:39
separation ofthe methylthio indenefrom the guinea
metabolites
Compounds
of indene
from
were separated
and diol metabolites pig.
the guinea
pig; B, control
as TMS-derivatives;
are the methylthio compounds I and peaks III and IV are the indane-l,2-diols 294 = M).
2.6, 6.0 4.3, 6.6 4.9,5.4 9.8, 10.0
was due to the benzylic methine proton. Irradiation of the multiplet at 4.46 ppm resulted in a singlet at 4.03 ppm and two doublets at 3.29 and 2.84 ppm, which showed that the signal at 4.46 ppm arose from the secondary methine proton of isomer I. A heteronuclear proton-carbon chemical shift correlation NMR spectrum (1 3) of isomer I was then obtained (fig. 3). This twodimensional spectrum shows that the ‘3C-signal of the carbon attached to the methylthio moiety (57.4 ppm) correlates with the benzylic
200
7:23
5. GC-MS
A, urinary
IV
Ill
14.3 100.0 12.5 100.0
oxide
150
6:18
.
i;zg/kg
Indene
100
5:12
Diol
I Indenea Indenea Indeneoxide’ Indene oxidea
FIG.
.
Methlth,o .
so 4:07
peaks
II, respectively (ml: 252 III and IV, respectively
of
urine I and II =
(m/:
diols III and IV, were also observed in both types of reaction mixtures. The overall conversion of indene oxide to isomer I by the sulfonium pathway was shown to proceed readily. Isolation of isomer I directly from the reaction ofN-acetyl-L-methionine and indene oxide, without HPLC purification ofthe sulfonium intermediate, yielded 19% of the desired product (fig. 4). Similar yields (23-36%) were obtained by Dekker (6) for the related reaction of 1,3-dichloropropene and methionine. The formation of2-hydroxy-l-methylthioindane was found to be a significant route of metabolism in the guinea pig for indene, indene oxide, and conjugates of the glutathione pathway (table 1). The methylthio isomers (I and II), identified as urinary metabolites of indene, comprised a total of 6.5-9.6% of the administered dose (fig. 5). The formation ofisomer I from indene oxide was found to occur with 4-14% ofthe dose being converted to this metabolite. Administration of indene oxide to the rat yielded only small amounts of isomer I (0.3%). The cis- and trans-indane-1,2-diols were significant metabolites of indene and its oxide in both the rat and guinea pig; the trans isomer (IV) predominated in both species. 1-GLU and 1-MER were prepared with the procedure develo_ for the corresponding styrene conjugates (14). Administralion of 1-GLU (1 1.6 and 18.5 mg/kg) to pairs of guinea pigs afforded isomerl as a significant urinary metabolite(9.6-l 1.7%). 1-MER was also metabolized to isomer I in the guinea pig. Neither isomer of indane-l,2-diol was detected as a metabolite of the two indene oxide conjugates.
gave
primarily the hydroxy(methylthio)indane isomer I, which mdicates nucleophilic attack of methionine at the 1-position of indene oxide. The minor methylthio isomer II, as well as the
Discussion Administration
cant
amounts
of indene to the guinea pig resulted in signifiof the methylthio metabolites I and II; the major
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2015
t’t/lllt/li(
()/lfl
,
230
TABLE J-or?nalion
2
I
180
inethvlthioinda,ie
A molecular
.
METHYLTHIO metabolite
the
was
methylthio
intermediate
A probable be through
I. Work
with
moiety
arises
related
compounds
from
suggested
that
of a reactive with endogenous sulthydryl compounds (3, 4, 7). route of methylthio formation for indene then would indene oxide, a major oxidative metabolite in vivo
reaction
(9) and
in vitro
showed
large
isomer
II,
fraction
of the lower dose of indene oxide was converted to I than was the higher dose (14% vs. 4%), which suggests
isomer
(1 5). Subsequent
the
METABOLITES
of isomer
I, along
urinary
metabolites
in
as
a possible
experiments
amounts
saturation
of
one
or
pathway at the higher dose. The origin of the methylthio investigation
by
several
with
with
the
steps
moiety
has
Tateishi
pig.
in
this
been
oxide
amounts
guinea
more
groups.
indene
small
of
A
larger
metabolic
the subject
of
( 1) identified
et a!.
a
lism
of the
vivo,
also yields
pre-mercapturic
acid
and
gate
C-S /3-lyase,
kidney
(19), Subsequent
of
and
intestinal
flora
methylation
(16) by
the
sibility
that
conjugates tabolized pig.
glutathione the
pathway
methylthio
methylthio
for cysteine
(16)
yield
an
such
(8, 9) we
rat
in
intermediate as thiol
investigated
is formed
meth-
metabolite. is metabolized
I and
1-GLU and of metabolite
metabolite
followed by S-methylation, to the cysteine conjugate, lyase
in the
the methylthio via its oxide,
metabolites
of this pathway. Both to significant amounts
If this
to
enzymes
yltransferase (20) would then yield With the knowledge that indene, through
naphthalene,
metabolites (1 7). These mercapturic conjugates may be cleaved by cysteine conjuan enzyme present in mammalian liver (18),
cysteine
thiol.
acid
methylthio
II
the
pos-
arose
from
1-MER were meI in the guinea via
a C-S
lyase,
we presume that 1-GLU is hydrolyzed based on the known specificity of the or mercapturic
acid
conjugates
(1).
The
quantities of the methylthio metabolites of indene oxide varied significantly between species. Whereas 4-14% of a dose of the oxide was converted to isomer I in the guinea pig, only small
amounts
(0.3%)
of this
compound
were
seen
in the
rat.
for the formation of the analogous styrene oxide metabolites. The guinea pig and rat converted 6.57.5% and 1-2%, respectively, of a dose of styrene oxide (100 mg/kg) to two hydroxy(methylthio)phenylethanes (7). Inasmuch as the guinea pig has been found to N-acetylate cysteine conjugates less readily than the rat (2 1), a larger fraction of the compounds administered may be present as cysteine conjugates in the guinea pig. These higher levels could then result in greater methylthio metabolite formation if the C-S lyase responsible for Similar
this
results
seen
were
conversion
is specific
for S-substituted
cysteine
conjugates.
In conclusion, we have found that methylthio formation significant metabolic pathway for indenc and its oxide in guinea pig, but not in the rat. Work with glutathionc mercapturic acid conjugates of indene oxide shows that metabolic route is a continuation of the glutathione pathway. Acknowledgments. lin
(Baylor
spectra
and
ing the
NMR
College
The of
authors
Medicine)
Dr. G. E. Martin
wish for
(University
to thank obtaining
Ms. Jean the
of Houston)
is a the and this
Now-
FAB-mass
246 (1970).
H. Tomisawa, S. lchihara. H. Fukazawa. and M. Tateishi: A C-S bond cleavage enzyme of cysteine conjugates in intestinal microorganism. Bioe/u’m. P/iarmaeol. 31, 2 1 37-2 140 (1982). I 7. J. Bakke, C. Struble. i-A. Gustafsson. and B. Gustafsson: Catabolism of premercapturic acid pathway metabolites of naphthalene to naphthols and methlthio-containing metabolites in rats. Proc. 16.
S. Suzuki.
Nii/.
.l((l(/.
.S(.i.
LS..
I. 82,
668-671
(1985).
18. P. M. Anderson and M. 0. Schultze: Cleavage of S-(l,2-dichlorovinyl)-i.-cystcinc by an enzyme of bovine origin. Arc/i. Biochem. Th0P11.1 I I I, 593-602 (1965). 19. M. Tatcishi and H. Shimizu: Cysteine conjugate /3-lyase. In “Enzymatic Basis of Detoxication” (W. B. Jakoby, ed), vol. II, pp. 121I 30. Academic Press, New York, 1980. 20. R. A. Keith, I. Jardine, A. Kerremans, and R. M. Weinshilboum:
Human
for obtain-
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I . M. Tateishi. S. Suzuki, and H. Shimizu: The metabolism of bromazepam in the rat-identification of mercapturic acid and its
INDENE
21.
erythrocyte
membrane
thiol
methyltransferase.
S-Meth-
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of in
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mercapturic acid of bromazepam and found that it is metabolized to the corresponding methylthiobromazepam in the presence of the rat liver 9000g supernatant. The cysteine conjugate of bromobenzene is converted to p-bromothiophenol by microorganisms present in the large intestine of the rat (16). Metabo-
OF