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-

References

101

conversion in vitro to methylthio-bromazepam. Biochem. Pharmacol. 27, 809-810 (1978). 2. T. Mio, K. Sumino, and T. Mizutani: Sulfur-containing metabolites of 2,5,2’,S’-tetrachlorobiphenyl, a major component of commercial PCB’s. Chem. Pharm. Bull. (Tokyo) 24, 1958-1960(1976). 3. W. G. Stillwell, 0. J. Bouwsma, J.-P. Thenot, M. G. Horning, 0. W. Griffin, K. Ishikawa, and M. Takaku: Methylthio metabolites of naphthalene excreted by the rat. Res. Commun. Chem. Pathol. Pharmacol. 20, 509-530 ( I978). 4. T. Mizutani, K. Yamamoto, and K. Tajima: Bromo(methylthio)benzenes and related sulfur-containing compounds: minor urinary metabolites of bromobenzene in rats. Biochem. Biophvs. Res. Commun. 82, 805-8 10 (1978). 5. D. H. Chatfield and W. H. Hunter: The metabolism of acetamidothiazoles in the rat. Biochem. J. 134, 879-884 ( I 973). 6. W. H. Dekker: 3-Chloroallyl methyl sulfide, a product from the reaction of I ,3-dichloropropene and biological materials. Meded. Fac. Landhouwwet. Rijksuniv. Gent 37, 865-868 (1972). 7. K. Nakatsu, S. Hugenroth, L-S. Sheng, E. C. Horning. and M. 0. Horning: Metabolism of styrene oxide in the rat and guinea pig. Drug Metab. Dispos. I 1, 463-470 (1983). 8. T. J. R. Francis, R. J. Bick, P. Callaghan, and R. P. Hopkins: The role of 1,2-epoxyindene in the metabolism of indene by rat liver fractions. Biochem. Soc. Trans. 3, 1244-1246 (1975). 9. F. A. Kerdel, R. J. Bick, R. P. Hopkins, and P. Callaghan: The role of 1,2-epoxyindene in the metabolism of indene in vivo. Biochein. Soc. Trans. 6, 785-787 (1978). 10. C. J. W. Brooks and L. Young: Biochemical studies oftoxic agents. 9. The metabolic conversion of indene into cis- and trans-indaneI ,2-diol. Biochem. J. 63, 264-269 (1956). I 1. A. J. Ryan and J. R. Bend: The metabolism ofstyrene oxide in the isolated perfused rat liver. Identification and quantitation of metabolites. Drug Metab. Dispos. 5, 363-367 ( I 977). 12. L-S. Sheng, E. C. Horning, and M. 0. Horning: Synthesis and elimination reactions of methylsulfonium ions formed from st rene oxide and methylthio compounds related to methionine and cysteine. Drug Metab. Dispos. I 2, 297-303 ( I 984). I 3. W. P. Aue. E. Bartholdi. and R. R. Ernst: Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. C/tern. P/n’s.64, 2229-2246 (1976). 14. B. Yagen, 0. L. Foureman, Z. Ben-Zui. A. J. Ryan. 0. Hernandez. R. I-I. Cox and J. R. Bend: The metabolism and excretion of ‘3Cstyrene oxide-glutathione adducts administered to the winter flounder, Pseudopleuronectes atnerieanu.s, a marine teleost. Drug Metab. Dispos. 12, 389-395 (1984). I 5. K. C. Leibman and E. Ortiz: Epoxide intermediates in microsomal oxidation ofolefins to glycols. J. P/iar,navl. Evp Tlu’r. 173, 242-

spectra.

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-

ylation of captropril, N-acetylcysteine and 7a-thio-spirolactone. Drug Metab. Dispos. I 2, 7 1 7-724 ( I 984). H. 0. Bray. T. J. Franklin. and S. P. James: The formation mercapturic acids. 3. N-Acetylation of S-substituted cysteines the rabbit, rat and guinea pig. Biochein. .1. 73, 465-473 (1959).

of in

Downloaded from dmd.aspetjournals.org at ASPET Journals on May 5, 2015

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

Hypertrophic cardiomyopathy: a review.

Hypertrophic cardiomyopathy (HCM) is a global disease with cases reported in all continents, affecting people of both genders and of various racial an...
1MB Sizes 6 Downloads 23 Views