Metabolic inactivation of five glycidyl ethers in lung and liver of humans, rats and mice in vitro

Article abstract of DOI:10.1080/004982500237497

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C₁₂GE) and o-cresol (o-CGE), has been studied in subcellular fractions of human, C₃H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C₁₂GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.

Full text of DOI:10.1080/004982500237497

xenobiotica
vol
no
. 30, . 5, 485±502
, 2000,
Metabolic inactivation of Ž ve glycidyl ethers in lung
and liver of humans, rats and mice
in vitro
P. J. BOOGAARD*, K. P. DE KLOE, J. BIERAU, G. KUIKEN,
P. E. D. BORKULO and N. J. VAN SITTERT
Department of Molecular Toxicology, Shell Research and Technology Center,
Amsterdam, Shell International Chemicals BV, PO Box 38000, 1030 BN Amsterdam,
The Netherlands
Received 23 July 1999
-
1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short term in
vitro tests and consequently GE are considered to be potentially mutagenic in vivo.
å
However, GE may be metabolically inactivated in the body by two di erent enzymatic
routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione
-
(GSH) catalysed by glutathione S transferase (GST) or hydrolysis of the epoxide moiety
catalysed by epoxide hydrolase (EH).
å
2. The metabolic inactivation of Ž ve di erent GE, the diglycidyl ethers of bisphenol A
-
-
-
-
(BADGE), 4,49 dihydroxy 3,39,5,59 tetramethylbiphenyl (Epikote YX4000) and 1,6
-
-
-
hexanediol (HDDGE) and the GE of 1 dodecanol (C GE) and o cresol(o CGE), has been
studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung.
"#
3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were
rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The
aromatic GE were very good substrates for EH. In general, microsomal EH is more
æ
æ
e
cient than cytosolic EH in hydrolysis of GE, and human microsomes are more e cient
than rodent microsomes.
-
-
4. The more water soluble GE, o CGE and HDDGE, were good substrates for GST
whereas the more lipophilic GE, YX4000 and C GE, were poor substrates for GST. In
"#
æ
general, rodents are more e cient in GSH conjugation of GE than humans.
5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the
æ
least e ciently inactivated of the Ž ve GE under study. For the other three GE no general
æ
trend was observed: the relative e ciency of inactivation varied with organ and species.
6. The large variation in metabolism observed with Ž ve representativeGE indicate that
GE have variable individual properties and should not be considered as a single,
homogenous class of compounds.
Introduction
Glycidyl ethers (GE) form an important group of chemicals used widely and in
a large variety in the manufacture of epoxy resin systems employed in adhesives,
coatings, plastics, electronics and structural composites. Some glycidyl compounds
have been reported to show direct mutagenic properties in bacteria and in a small
et al
. 1992). For
number of examples to cause tumours in animal studies (Gardiner
example, phenyl glycidyl ether (PGE) and resorcinol diglycidyl ether have been
classiŽ ed by IARC as Class 2B carcinogens (‘possibly carcinogenic to humans’)
(IARC 1985, 1989, 1999a, b). Other glycidyl ethers are under scrutiny because of
their structural relationship to this glycidyl ether.
in vivo
On the basis of previous metabolic studies
et al et al
on glycidylcompounds(Climie
. 1981a, b, Boogaard . 1999) it is expected, however, that most GE would
be rapidly and eåectively detoxiŽ ed by epoxide hydrolysis or by conjugation with
-
* Author for correspondence; e mail: Peter.J.Boogaard!opc.shell.com
}
-
-
ISSN 0049 8254 print ISSN 1366 5928 online ’ 2000 Taylor & Francis Ltd
Xenobiotica
}}
}
http: www.tandf.co.uk journals

P J Boogaard
. .
486
et al.
Figure 1. Metabolic detoxiŽ cation pathways for glycidyl ethers.
glutathione (GSH) (Ž gure 1). Therefore, comparative metabolism studies on GE of
diåerent classes were carried out to identify the role of these detoxiŽ cation reactions
in the overall toxicology, such that glycidyl ethers which are readily susceptible to
hydrolytic or conjugative detoxiŽ cation reactions can be clearly diåerentiated.
Moreover, it is essential to generate data to substantiate the view that glycidyl
compounds should not be considered as a single class of compounds but that
-
susceptibility to metabolic hydrolysis and intrinsic and enzyme catalysed con
jugation with glutathione should be taken into account.
One of the primary portals of entry for GE is the lungs through inhalation of
aerosols or vapours. Nevertheless, hepatic metabolism was also considered relevant
for two reasons. First, residues of GE may be present in foodstuås and ingestion of
GE might occur, either directly or from swallowing material deposited in the
airways after inhalation. In these cases hepatic metabolism may play a signiŽ cant
role. Second, the metabolic capacity of the liver is considerable and will probably be
the single most important factor in the overall metabolism of GE that became
systemically available, regardless of the way it entered the body. The skin may also
be an important route of exposure for GE through direct dermal contact.
Percutaneous penetration and metabolism of the Ž ve selected GE has also been
et al
. (2000).
studied and will be reported in Boogaard
The overall objective of this study was generation of mechanistic data on the
detoxifying metabolism of a series of GE to assist its appropriate classiŽ cation and
-
labelling. Representatives of four classes of GE (mono and difunctional aromatic
-
GE and mono and difunctional aliphatic GE) were selected and synthesized and
S-
their rates of epoxide hydrolase (EH) catalysed hydrolysis and glutathione
-
transferase (GST) catalysed conjugation with GSH were studied in subcellular
fractions of liver and lung of human, Fisher 344 rat and C3H mouse.
Materials and methods
Chemicals
}
"%
-
-
[U C] epichlorohydrin, radiopurity & 97%, speciŽ c activity 30 mCi mmol, was from ChemSyn
$
- -
-
Laboratories (Lenexa, KS, USA), and [glycine 2 H] glutathione, radiopurity & 97%, speciŽ c activity
}
"%
-
-
-
-
44.8 mCi mmol, from NEN Life Science Products (Boston, MA, USA). [Ring U C] bis phenol A
}
diglycidyl ether, radiopurity & 98 %, speciŽ c activity 11 mCi mmol, was a gift from Dr John Waechter
(Dow Chemical Co., Midland, MI, USA). Re crystallized BADGE (chemical purity 99%) and its bis
"
-
-
"
-
diol (bis phenol A diglycerol ether; chemical purity
98%) were from the department of Basic &
-
-
-
-
Exploratory Research (Shell Research & Technology Centre, Amsterdam). 1,2 Epoxy 3,3,3 trichloro

Metabolic inactivation of glycidyl ethers
487
Figure 2. Structures of synthesized chemicals.
Table 1. Characteristics of human liver donors.
}
Protein (mg ml)
}
Medication
Age
No. Sex Race
(years) CODa illnessesb
Alcohol Smoker Microsomes Cytosol
1
F
Caucasian
49
ICH premarin,
ditropan,
no
no
20
18.8
}
desyrel HTN
}
2
3
F
F
Caucasian
Caucasian
42
53
ICH clonidine HTN
no
no
no
no
20
20
20
20
}
ICH oestrogen
CNS tumor
CHI none
4
5
6
M
M
M
Caucasian
Caucasian
Caucasian
32
39
51
yes
yes
no
no
no
no
20
20
20
20
19.8
20
ICH none
CHI HTN
a
-
Cause of death (COD): ICH, intracranial haemorrhage; CHI: closed head injury.
b HTN, hypertension; CNS, central nervous system.
-
propane (TCPO), reduced glutathione (GSH), oxidized glutathione (GSSG) and micro protein
-
determination kits were from Sigma Chemical Co. (St Louis, MO, USA). 4 Phenylchalcone oxide
-
(4 PCO) was from ICN Biomedicals BV (Zoetermeer, The Netherlands). Solvents were of the highest
grades available. All other chemicals used were from Aldrich and were at least reagentgrade with a purity
"
95%.
Reference materials
The glycidyl and glycerol ethers of o cresol and 1 dodecanol as well as the diglycidyl and diglycerol
-
-
-
-
-
-
ethers of 1,6 hexanediol, 4,49 dihydroxy 3,39,5,59 tetramethylbiphenyl and diphenylol propane were
"%
-
synthesized from the alcohols and epichlorohydrin (ECH) (Ž gure 2). The corresponding C labelled

P J Boogaard
. . et al.
488
"%
-
-
-
compounds were synthesized from the alcohols and [U C] ECH, diluted with non labelled ECH to a
}
speciŽ c activity of 0.2 mCi mmol. The aromatic alcohols were converted to the corresponding glycidyl
ethers using sodium hydroxide and aliphatic alcohols using tin(IV) chloride as catalyst. All compounds
"
"
"$
-
-
-
were identiŽ ed by
H
and C NMR and mass spectrometry. Chemical purities (from 92 to 99%)
"
"
-
were determined using GC MS or LC MS. Radiopurities (from 95 to 98%) were determined by
-
GC and LC with on line radiodetection. Details of synthesis and analysis are in van Elburg et al. (2000).
Biological materials
å
Human organs. Human liver and lung microsomes, prepared by standard di erential centrifugation
methods (Boogaard et al. 1996), were from the Human Cell Culture Center (Laurel, MD, USA). Liver
and lung donor characteristics are listed in tables 1 and 2 respectively.
C
C
Animals. Male Fisher 344 rats ( 250 g) and male C3H mice ( 25 g) were from the Central
Laboratories for the Blood Banks (CLB) (Amsterdam, The Netherlands). Animals were kept on a 12 h
}
-
-
light dark cycle in humidity (60 15% relative humidity) and temperature (22 2 C) controlled
Ê
6
6
-
-
mass air displacement rooms and following an acclimation of 1 week were used as the source for skin,
-
liver and lung tissue. A standard rodent diet (VRF I, Broekman Instituut (Charles River Laboratories),
-
Someren, The Netherlands) and de ionized water were supplied ad libitum.
Subcellular tissue fractions
Rodents were anaesthetized with sodium pentobarbital, the abdomen was opened and the abdominal
-
-
å
aorta cut. The animals were subsequently perfused in situ with ice cold isotonic 0.05 m TRIS bu ered
(pH 7.40) 0.15 m potassium chloride solution. Livers and lungs were excized, weighed and placed on ice.
The lungs were minced with scissors and pulverized in liquid nitrogen using a hammer mill (6700
}
Freezer Mill, Glen Creston, Inc., Stanmore, UK). Livers were only minced with scissors.Lung powder
-
å
and minced livers were homogenized in TRIS bu ered (pH 7.40) isotonic KCl solution with six passes
(1100 rpm) of a Te on glass homogenizer (Braun). Homogenates were centrifuged at 10 000g at 4 C for
±
Ê
20 min, the lipid layer was removed and the supernatant was centrifuged at 105000g at 4 C for 60 min.
The supernatant (cytosolic fraction) was stored frozen at 80 C. The pellet was dispersed with four
passes of a Te on glass homogenizer in 0.05 m TRIS bu er (pH 7.40) containing 0.25 m sucrose and 1
Ê
2
Ê
å
±
mm EDTA and centrifuged again at 105000g at 4 C for 60 min. The supernatant was discarded and the
Ê
å
pellet resuspended with four passes of a Te on glass homogenizer in 0.1 m potassium phosphate bu er
±
(pH 7.40) containing 0.25 m sucrose (microsomal fraction). The microsomes were stored at 80 C.
±
Ê
Subcellular fractionswere used within 6 months of preparation. No apparent loss of activity was observed
during this period as determined by assaying epoxide hydrolysis and GSH conjugation of standard
substrates (Boogaard and Bond 1996, Boogaard et al. 1996).
Protein concentrations of all lung and liver subcellular preparations of rodents were assayedusing the
-
-
modiŽ ed micro Lowry assay for total micro protein. Human liver and lung subcellular preparations
-
were assayed for total micro protein using the Coomassie blue method (all Sigma).
Analyses
Liquid scintillation counting(LSC). Radioactivity in solutions was measured using TriCarb 2200CA
liquid scintillation counters (Canberra Packard, Groningen, The Netherlands) in antistatic scintillation
vials after mixing with 5 10 vols Ultima Gold Scintillation cocktail (Canberra Packard). The machine
±
$
"%
was calibrated using commercial H and C internal standard kits for organic solvents (Wallac, Turku,
Finland). The calibration was checked daily by counting a set of quenched standards commercially
æ
prepared in sealed glass vials. Counting e ciency was determined using the spectralindex of the internal
standard (SIE) and cpm were automatically transformedto dpm. Samples were correctedfor background
!
æ
and counted for periods su ciently long to reach a relative SD 0.02.
}
Capillary zone electrophoresis (CZE). CZE was carried out on a Beckman P ACE 2100 system
equipped with a UV absorbance detector, set at k 214 nm. The temperature of the capillary was
5
6
controlled by liquid cooling and was maintained at 23 1 C. Data were recordedwith Beckman System
Ê
-
3
Gold software. Beckman pretreated fused silica capillary cartridges, 50 cm 75 lm (internal diameter)
-
å
å
were used for separation using a 12.5 mm Borax HCl bu er, pH 8.35, as separation bu er. The
capillaries were subsequently rinsed with 0.1 m NaOH for 30 s, with puriŽ ed water for 30 s, and with
å
separation bu er for 1 min, samples were injected by application of high pressure for 1 2 s, all
±
-
separations were carried out at 19 kV in 16.5 min. The GSH conjugates of BADGE, o CGE and
HDDGE had retentions of 12.7, 13.0 and 13.2 min respectively, while GSH and GSSG had retentions
of 13.7 and 14.2 min respectively. Initially, aspartame (retention 12.7 min) was chosen as internal
-
standard based on the structural similarity to the GSH conjugate of o CGE. However, it appeared
-
- -
unstable in aqueous solution as it demethylated over time. l Asp l Phe, in contrast, was stable and
appearedas a suitable internal standard(retention15.8 min) for the determination of the GSH conjugates

Metabolic inactivation of glycidyl ethers
489
Table 2. Characteristics of human lung donors.
}
Protein (mg ml)
}
Medication
Age
No. Sex Race
(years) CODa illnessesb
Alcohol Smoker Microsomes Cytosol
1
2
F
F
Caucasian
Caucasian
35
66
anoxia none
yes
no
yes
no
17.5
10
17.7
8.4
}
ICH
HTN medication
HTN, KS
cannabis, Prozac
anorexia
}
3
F
Caucasian
17
CHI
yes
yes
20
7.7
4
5
M
M
Caucasian
Caucasian
43
56
ICH
ICH
none
coumadin, aterol,
social
social
noc
no
15
20
18.9
14.5
}
-
K du, prenivil HD
}
6
M
Caucasian
59
SDH dilantin HTN
social
no
10
3.4
a Cause of death (COD): ICH, intracranial haemorrhage; SDH, subdural hematoma.
b HTN, hypertension; KS, kidney stones; HD, heart disease.
c Stopped smoking 9 years ago.
-
-
- -
of other GE as well. Several N benzoylated amino acids, such as N benzoyl l methionine (retention
-
-
13.9 min) and N benzoyl histidine (retention 13.7 min) were tested as external standards to correct for
å
di erences in injection volume. Based on a retention of 13.8 min and the large molecular extinction
æ
-
- -
coe cient, N benzoyl l Phe was selected as external standard.
Typical injection volumes for CZE were 9 nl, which is far too small to calibrate the analyses with
-
radiolabelled referencematerial used for calibration of the EH catalysed metabolism. As it was desirable
to calibrate all analytical methods with the same reference material, all radiolabelled GE were incubated
with mouse hepatic cytosol and GSH. The reaction was stopped by addition of methanol and following
precipitation of the protein, the supernatant was serially diluted to obtain a series of solutions with
å
di erent concentrations of GE. These solutions were subsequently analysed for the parent compound
and its GSH conjugate both by HPLC with LSC and by CZE to enable calibration of the latter method
of analysis.
-
High performance liquid chromatography (HPLC). HPLC analyses were carried out using various
-
liquid chromatographs. For analysis of radiolabelled compounds, a Ramona D or 2000 on line
radioactivity detector (Raytest, Straubenhardt, Germany), both equipped with 500 ll solid scintillant
cells, were used. In addition, since the total radioactivity in individual peaks was in most cases below the
-
detection limit of the on line detectors, fractions were collected using fraction collectors. The
radioactivity in the collected fractions was determined by LSC. HPLC analyses were carried using the
following equipment and conditions.
System A. Apparatus: HP1050 liquid chromatograph with UV detection (k 230 nm) (Hewlett
5
3
Packard,Amstelveen, The Netherlands); column: Phenomenex Jupiter 300 A, 5 lm ODS, 250 4.6 mm
(Phenomenex, Torrance, CA, USA) with HiChrom security guard column NC120 5C18 10C
±
±
}
(HiChrom, Reading, UK); eluent: puriŽ ed water (A) and methanol (B);  ow: 1.0 ml min; gradient:
0 18 min, linear increase from 25 to 92.5% B; 18 20 min, 92.5% B; 20 22 min: linear increase from
±
±
±
92.5 to 100% B, 30 35 min linear decrease from 100 to 25% B.
±
System B. Apparatus: HP1090 liquid chromatograph with UV detection (k 230 nm); column:
5
3
-
Waters Novapak C18, 3 lm ODS, 15 3.9 mm (Waters, Etten Leur, The Netherlands), with
3
Phenomenex Security Guard (widepore 4 3.0 mm ODS cartridge); eluent: puriŽ ed water (A) and
}
acetonitrile (B);  ow: 1 ml min; gradient: 0 31 min, linear increase from 25 to 48.5% B; 31 35 min,
±
±
linear increase from 48.5 to 100% B; 35 40 min: linear decrease from 100 to 25% B; 40 45 min:
±
±
25% B.
System C. Apparatus: HP1100 liquid chromatograph with UV detection (k 230 nm) (Hewlett
5
3
Packard); column: Phenomenex Jupiter 300 A, 5 lm ODS, 250 4.6 mm, with Phenomenex Security
3
Guard (widepore 4 3.0 mm ODS cartridge); eluent : puriŽ ed water (A) and acetonitrile (B);  ow:
}
1.0 ml min; gradient: 0 31 min, linear increase from 25 to 48.5 % B; 31 35 min, linear increase from
±
±
48.5 to 100% B; 35 40 min, linear decrease from 100 to 25% B; 40 45 min 25% B.
±
±
System D. Apparatus: HP1100 liquid chromatograph with UV detection (k 230 nm) (Hewlett
5
3
Packard); column: Phenomenex Jupiter 300 A, 5 lm ODS, 250 4.6 mm, with Phenomenex Security
3
Guard (widepore 4 3.0 mm ODS cartridge); eluent : puriŽ ed water (A) and acetonitrile (B);  ow:

P J Boogaard
. .
490
et al.
}
1.0 ml min; gradient: 0 15 min, linear increase from 0 to 30% B; 15 25 min, linear increase from 30 to
±
±
45% B; 25 30 min, linear increase from 45 to 100% B; 30 33 min, 100% B; 33 35 min linear decrease
±
±
±
from 100 to 0% B.
System E. Apparatus: HP1100 liquid chromatograph with UV detection (k 230 nm) (Hewlett
5
3
Packard); column: Phenomenex Jupiter 300 A, 5 lm ODS, 250 4.6 mm, with Phenomenex Security
3
Guard (widepore 4 3.0 mm ODS cartridge); eluent : puriŽ ed water (A) and acetonitrile (B);  ow:
}
1.0 ml min; gradient: 0 20 min, linear increase from 25 to 40% B; 20 21 min, linear increase from 40
±
±
to 100% B, 21 25.5 min 100% B; 25.5 26.5 min, linear decrease from 100 to 25% B; 26.5 30 min
±
±
±
25% B.
System F. Apparatus: Knauer HPLC Pumps 64, controlled by a Programmer 50 and equipped with a
Dynamic Mixing Chamber and a Variable UV Detector (set at k 230 nm) (Herbert Knauer, Berlin,
5
3
Germany); column: Phenomenex Jupiter 300 A, 5 lm ODS, 250 4.6 mm, with HiChrom security
}
guard column NC120 5C18 10C; eluent: PuriŽ ed water (A) and acetonitrile (B);  ow: 1.0 ml min;
±
±
gradient: 0 25 min, linear increase from 25 to 100% B; 25 32 min, 100% B; 32 37min: linear decrease
±
±
±
from 100 to 25% B.
-
-
-
Gas chromatography mass spectrometry (GC MS). GC MS analyses were carried out using a HP 6890
-
-
GC equipped with auto injector and a HP 5973 mass selective detector on a HP 5 MS, fused silica
3
column of 30 m 0.25 mm (internal diameter) with a Ž lm thickness of 0.25 lm and a phase ratio of 250.
}
C
The mobile phase was He at a constant  ow of 1.0 ml min (pressure 7.7 psi). The injector was kept at
}
275 C and 1 ll split splitless injections were made. The following temperature programme was used:
50 C for 1 min, then at 10 C min to 200 C, maintained at 200 C for 10 min; then second ramp
(10 C min) to 250 C, maintained at 250 C for 10 min. Temperature transfer line was kept at 250 C.
The mass detector was used at electron impact mode; the quadrupole was kept at 106 C and the source
at 230 C. The ionization potential was 70 eV and the multiplier voltage was set at the autotune voltage
plus 400 eV. The data collection was either in scan mode from m e 20 to 500 or, for quantitation, by single
ion monitoring (100 ms dwell time) of the following fragments: m e 111 and 197 (fragments of C GE),
Ê
}
Ê
Ê
Ê
Ê
}
Ê
Ê
Ê
Ê
Ê
Ê
}
}
"#
}
m e 199 and 219 (fragments of the diol of C GE).
"#
-
-
-
Liquid chromatography mass spectrometry(LC MS). LC MS analyses were performed using system A
on a HP 1050 liquid chromatograph coupled to a Micromass Quattro quadrupole mass spectrometer.
-
Electrospraywas used as the LC MS interfacing and ionization technique (positive ions). The scan range
}
was 100 1500 Dalton with 0.5 scan s.
±
GSH conjugation
Uncatalysed chemical reaction. The chemical reaction of the GE with GSH was studied with both
-
labelled and non labelled GE. Each individual, unlabelled GE was dissolved in DMSO at a concentration
$
-
of 20 mm. After dissolution an equal volume of a 20 mm solution of H GSH, freshly prepared in
å
deoxygenated 100 mm phosphate bu er, pH 7.4, was added to a Ž nal volume of 5 ml. The mixture was
kept at room temperature in an amber vial for 18 h and aliquots were analysed by HPLC (system A, see
"%
-
-
above) and CZE. C labelled GE were allowed to react with non labelled GSH similarly and analysed
by HPLC and CZE.
Enzymaticreaction for GSH conjugation. Individual GE under study were dissolved in acetone,aliquots
were transferred into 1.5 ml vials and the acetone was carefully evaporated with a gentle stream of
å
-
- -
nitrogen. Phosphate bu er (0.1 m, pH 7.40), containing l Asp l Phe as internal standard, was
å
deoxygenated with He for 10 min. GSH was dissolved in the deoxygenated phosphate bu er. For
-
cytosolicincubations, an aliquot of 4 PCO, an inhibitor of cytosolic EH (Mullin and Hammock1982)was
dissolved in acetone and transferred into a 2.0 ml vial and the acetone was evaporated under a gentle
å
stream of nitrogen. Cytosol was diluted with the deoxygenatedphosphate bu er and transferredinto the
-
vial with 4 PCO. For microsomal incubations, microsomes were diluted with the deoxygenated
å
phosphate bu er and transferred into 2.0 ml vials. TCPO, an inhibitor of microsomal EH (Guest and
!
}
Dent 1980), dissolved in DMSO was added in a small volume (concentration of DMSO was 5% v v).
C
The mixtures of subcellular protein and inhibitors were incubated at 37 C for 2 min and aliquots of
the GSH solution were added. The Ž nal concentrations of inhibitors, l Asp l Phe and GSH during the
Ê
-
- -
-
incubation were 50 lm, 1.5 mm and 10 mm respectively. After pre incubation for another 2 min, aliquots
of the subcellular protein mixtures were transferredto the vials with the GE. The Ž nal concentrations of
the GE were 0, 0.041, 0.12, 0.37, 1.11, 3.33 and 10.0 mm. Incubation was stopped by the addition of an
-
-
- -
equal volume of ice cold methanol (kept on dry ice), containing 127 lm N benzoyl l Phe, and the
precipitated protein pelleted by centrifugation (10 min, 2500g, 4 C). The supernatants were analysed by
Ê
CZE. Linearity of the GSH conjugation with protein concentration was tested by incubation during
}
10 min with varying amounts of liver cytosol protein ranging from 0.2 to 5.0 mg protein ml. Linearity

Metabolic inactivation of glycidyl ethers
491
with time was investigated by incubation during 0.5, 1, 2, 5, 10, 15, 20 and 30 min with a liver cytosol
}
protein concentration of 1 mg ml. Spontaneous reaction of GSH with GE or inhibitors was measured
from incubations without addition of cytosolic protein. All values for enzymatic reactions were corrected
for the measured uncatalysed chemical reaction.
Hydrolysis
-
Incubations were carried out with both microsomaland cytosolic fractions. Following pre incubation
of the subcellular fractions, the reaction was started by addition of the GE dissolved in DMSO. The Ž nal
concentrations of the GE ranged from 0.013 to 20.0 mm. The maximum concentration of DMSO in the
}
Ž nal incubation mixture was 5% v v.
For HPLC analysis, the incubation was stopped by addition of an equal volume of cold methanol
(kept on dry ice), mixing and precipitation of the denaturated protein by centrifugation (2400g, 15 min).
å
Various HPLC systems analysed the hydrolysis products of the di erent GE: BADGE was analysed
-
using system B, YX4000 was analysed using system C, HDDGE was analysed using system D, o CGE
was analysed using system E, and C GE was analysed using system F. Since the UV absorptions of
"#
HDDGE and C GE are not suitable for UV detection, hydrolysis of these compounds was studied using
"#
radiolabelled material and HPLC with fraction collection followed by LSC. Linearity of the hydrolytic
reactions with protein concentration was tested by incubation during 10 min with varying amounts of
liver microsomal protein. Linearity with time was investigated by incubation during 0.5, 1, 2, 5, 10, 15,
}
20 and 30 min with a liver microsome concentration of 1 mg protein ml.
}
æ
Estimation of the octanol water partition coe cient (P /
)
o w
æ
Log Po/w is in many casesan important determinant of enzyme a nity. For BADGE log P /
4.0 was
5
o w
determined by HPLC (Eadsforth1983).For the other GE, however, no log Po/w has been determined. For
these GE, log P / was estimated using the KOWWIN software package, v.1.57 (Syracuse Research
o w
Software, Whitstable, UK). The following were calculated: YX4000 log P /
5.2, C GE log P /
o w 5
"#
o w 5
-
5.0, BADGE log P /
3.8, o CGE log P /
2.2 and HDDGE log P /
0.84.
5
5
5
o w
o w
o w
Calculations and statistics
The formation of GSH conjugates or hydrolysis products in the incubation mixtures, after correction
-
for non enzymatic GSH conjugation, was Ž tted to the Michaelis Menten equation with parametervalue
±
optimization by the Levenberg Marquardt approximation, using the DeltaGraph software package
±
(v.4.0.5d; SPSS, Inc., Chicago, IL, USA). Experiments for the construction of Michaelis Menten plots
±
were done at least in duplicate for mouse and rat subcellular fractions, but if the results of duplicate
"
å
determinations di ered 20%, which sometimes happened with subcellular fractions with low activity,
additional series of incubations were performed to reduce the relative error associated with the Ž tted
}
parameters. Since V
and Km are interdependent variables, their ratio, Vmax Km , which may be
max
æ
interpreted as a measure of enzyme e ciency, was chosen as the best value for comparison of the
æ
e
ciency of the reactions between organs and species. In some cases no Michaelis Menten kinetics were
±
observed. In these instances the rate of the reaction was determined by linear regression analysis using
}
}
the least squares approximation. These reaction rates were expressed as ll min mg protein, units
}
identical to those in which V
Km is expressed, to allow easier comparison.
max
å
In vivo V was scaled from in vitro V and Km and by adjustment for the interspecies di erences
max
max
in microsomal and cytosolic protein concentrations and liver volumes (Boogaardand Bond 1996). In vivo
hepatic clearances were estimated by division of the calculated in vivo Vmax by the corresponding in vitro
Km for the enzymatic reaction. Average liver volumes for humans, mouse and rat were taken as 3.1, 5.4
and 3.7% of lean body weight respectively, and average lung volumes for humans, mice and rats were
0.65, 1.09 and 0.57% of lean body weight respectively (ILSI 1994). In this study, the cytosolic protein
}
}
content was assayed as 41.7, 72.6 and 50.1 mg g liver and 16.7, 45.1 and 10.4 mg g lung for human, rat
}
and mouse respectively, and the microsomal protein content was 16.7, 18.4 and 5.8 mg g liver and 2.7,
2.4 and 4.4 mg g lung for human, rat and mouse respectively.
}
Results
GSH conjugation
No detectable chemical or enzyme catalysed reaction of YX4000and C GE with
"#
GSH could be observed with either rat liver cytosolor microsomes(data not shown).
-
Both a small degree of chemical reaction and some inconsistent enzyme catalysed
GSH conjugation of BADGE was observed under the given experimental

P J Boogaard
. .
492
et al.
-
Table 3. Kinetic constants for enzymatic conjugation of o cresyl glycidyl ether
}
Km
ll min mg
V
Km
(mm)
V
max
max
}
}
}
}
Organ
Liver
Species
(nmol mg min)
n
R
human
rat
mouse
84.5
61.3
150
14.5
2.72
6.63
5.84
22.5
22.7
6
3
2
0.999
0.998
0.998
Lung
human
rat
mouse
50.1
29.0
29.5
20.9
5.79
3.95
2.40
5.01
7.45
6
2
2
0.997
0.993
0.998
}
n, Number of individuals animals.
æ
R, Correlation coe cient of the Ž tted curve.
-
Table 4. Kinetic constants for enzymatic conjugation of 1,6 hexanediol diglycidyl ether with
glutathione by liver and lung cytosol.
}
Km
ll min mg
Vmax
(nmol mg min)
Km
(mm)
V
max
}
}
}
}
Organ
Liver
Species
n
R
human
rat
mouse
nda
257
523
nda
5.69
13.4
4.95a
45.2
38.9
6
3
3
0.998a
0.937
0.998
Lung
human
rat
mouse
nda
98.6
129
nda
6.0
11.6
1.44a
16.4
11.0
6
3
3
0.996a
0.954
0.970
}
n, Number of individuals animals.
æ
R, Correlation coe cient of the Ž tted curve.
a No apparent Michaelis Menten kinetics, but enzymaticformationof glutathione conjugate appeared
±
}
proportional to the concentration of substrate under the incubation conditions. V
Km and R were
max
obtained by linear regression analysis.
o-
conditions. In contrast, CGE and HDDGE showed only a small but signiŽ cant
chemical reactivity towards GSH under the experimental conditions, but a rapid
enzyme catalysed reaction. The radiolabelled products formed during incubation of
$
o-
-
-
CGE and HDDGE with H GSH were identiŽ ed by LC MS as GSSG and as
o-
-
the GSH conjugates of CGE and HDDGE respectively. The GST catalysed
o-
reactions, for both CGE and HDDGE, were linear with time for at least up to
}
15 min and linear with protein concentrations up to 3 mg ml. All data are based on
}
10 min incubations with protein concentrations varying from 0.4 to 3.0 mg ml. The
parameters describing the above kinetic behaviour are tabulated in tables 3 and 4.
-
o-
The enzyme catalysed GSH conjugation of CGE, corrected for intrinsic chemical
reaction, was well described by Michaelis±Menten kinetics for both liver and lung
cytosol of rodents as well as humans. The hepatic GSH conjugation, expressed as
}
Vmax
K
m, was about equal in rodents but higher than in humans. Typical
o-
Michaelis±Menten plots for the GSH conjugation of CGE and HDDGE in the
presence of lung cytosol are represented in Ž gure 3. GSH conjugation in lung
"
"
cytosol was lower than in liver and decreased by: mouse rat human. The GSH
conjugation of HDDGE was less well described by Michaelis±Menten kinetics. In
human liver and lung cytosol, no Michaelis±Menten kinetics were observed, but the
increase in conjugation (corrected for the chemical reaction of HDDGEwith GSH)
was proportional to the concentration of HDDGE (Ž gure 3B), and a straight line
Ž tted better to the data obtained for the GSH conjugation of HDDGE in human
liver and lung cytosol than the rectangular hyperbola which would result from

Metabolic inactivation of glycidyl ethers
493
(a)
(b)
-
-
-
Figure 3. Enzymatic formation of the GSH conjugate of o cresyl glycidyl ether (o CGE) and 1,6
hexanediol diglycidyl ether (HDDGE) by human (D ), Fisher 344 rat (E ) and C3H mouse (+ )
lung cytosolic fractions. Data points represent means SE of six separate experiments with lung
6
-
cytosol from six individuals (three males, three females) or two (o CGE) or three (HDDGE)
-
separate experiments with lung cytosolfrom rodents. (a) o CGE. The lines are model simulations
#
for the Michaelis Menten equation Ž tted to the data points (R
human, rat and mouse lung respectively). (b) HDDGE. The lines for rat and mouse are model
simulations for the Michaelis Menten equation Ž tted to the data points (R
rat and mouse lung respectively). The data obtained for the GSH conjugation of HDDGE by
0.994, 0.986 and 0.996 for
±
5
#
0.910 and 0.941 for
±
5
human lung cytosol did not Ž t to the Michaelis Menten equation, but a linear increase of GSH
±
#
conjugates with the concentration HDDGE was found (R
0.992).
5
K
Michaelis±Menten kinetics. This might be caused by a low aænity (high m ) of
HDDGE for GST resulting in an apparently straight curve. Alternatively, the
-
bis-
conjugate, for which the peaks were not
formation of the mono conjugate and
well resolved at low concentration, might obscure the observation of Michaelis±
Menten kinetics. Again, the conjugation rates were higher in liver than in lung
fractions, and higher in rodents than in human. The GSH conjugation of HDDGE
o-
was, in contrast to CGE, slightly higher in rat than in mouse.
Hydrolysis
All Ž ve GE studied were rapidly hydrolysed to the corresponding (di)glycerol
ethers upon incubation with either cytosolic or microsomal fractions (Ž gure 4). The
o-
-
only exception was CGE for which no EH catalysed hydrolysis was observed with
rat lung cytosol (tables 5±9). The identity of the hydrolysis products formed was
-
conŽ rmed by co elution on HPLC with the synthesized reference compounds and
-
by comparison of the mass spectra obtained LC MS, with exception of C GE for
"#
-
-
which the mass spectrum was obtained by GC MS. The EH catalysed hydrolysis
was linear with time for at least 10 min and linear with protein concentrations up to
}
}
1.5 mg ml for liver and 3.0 mg ml for lung subcellular fractions. All data are based
}
on 10 min incubations and protein concentrations of 1.0 mg ml for liver and
}
}
Km
V
max
0.4±2.0 mg ml for lung preparations. In general, the
ratio for microsomal
EH was higher than for cytosolic EH. The only exception to this general observation
was seen with YX4000 and C GE in mouse liver. In these two cases, cytosolic
"#
}
V
max
K
m ratio, than microsomal
hydrolysis was more eåective, as determined by the

P J Boogaard
. .
494
et al.
(a)
(b)
(c)
(d)
(e)
Figure 4. Enzymatic hydrolysis of various glycidyl ethers by human (D ), Fisher 344 rat (E ) and C3H
mouse (+ ) lung microsomal fractions. Data points represent means SE of six separate
6
-
-
-
experiments with lung microsomes from six individuals (three males, three females) or two to
-
four separate experiments with lung microsomes from rodents. (a) YX4000 (4,49 dihydroxy
-
3,39,5,59 tetramethylbiphenyl diglycidyl ether). The lines represent model simulations for the
#
Michaelis Menten equation Ž tted to the data points (R
0.957, 0.943 and 0.998 for hydrolysis
±
5
by human, rat and mouse lung microsomes respectively). (b) BADGE (bisphenol A diglycidyl
ether).The lines represent model simulations for the Michaelis Menten equation Ž tted to the data
±

Metabolic inactivation of glycidyl ethers
495
-
-
-
Table 5. Apparent kinetic constants for enzymatic hydrolysis of YX4000 (4,49 dihydroxy 3,39,5,59
tetramethylbiphenyl diglycidyl ether) by liver and lung cytosol and microsomes.
}
Km
ll min mg
Vmax
(nmol mg min)
Km
(mm)
V
max
}
}
}
}
Organ
Liver
Fraction
cytosol
Species
n
R
human
rat
mouse
24.5
3.91
16.8
0.067
0.024
0.027
365
162
619
6
3
3
0.996
0.967
0.988
microsomes
cytosol
human
rat
mouse
133
30.6
12.0
0.18
0.064
0.12
731
475
102
6
3
3
0.984
0.987
0.972
Lung
human
rat
mouse
1.01
1.04
1.61
0.022
0.020
0.011
47
52
141
6
2
2
0.848
0.999
0.984
microsomes
human
rat
mouse
15.2
5.59
29.7
0.010
0.023
0.030
1524
241
978
6
3
4
0.978
0.970
0.999
}
n, number of individuals animals.
æ
R, correlation coe cient of the Ž tted curve.
Table 6. Apparent kinetic constants for enzymatic hydrolysis of BADGE by liver and lung cytosoland
microsomes.
}
Km
ll min mg
V
Km
(mm)
V
max
max
}
}
}
}
Organ
Liver
Fraction
cytosol
Species
(nmol mg min)
n
R
human
rat
mouse
26.1
12.2
52.1
0.22
0.37
0.27
117
32.6
198
4
6
3
0.999
0.948
0.987
microsomes
cytosol
human
rat
mouse
150
63.0
46.6
0.63
0.34
0.23
239
188
203
6
3
3
0.998
0.993
0.991
Lung
human
rat
mouse
2.90
2.02
3.45
0.16
0.26
0.25
18.7
7.80
13.6
6
3
5
0.942
0.994
0.940
microsomes
human
rat
mouse
12.5
14.5
19.3
0.08
0.38
0.36
157
38.1
53.4
6
3
2
0.993
0.950
0.985
}
n, number of individuals animals.
æ
R, correlation coe cient of the Ž tted curve.
}
Vmax
K
mwas highest for
hydrolysis. In general, for the Ž ve GE under study,
YX4000 hydrolysis and least for HDDGE hydrolysis. For the other three GE, no
}
V
max
Km
ratio for hydrolysis varied with organ and
general trend was observed: the
species. Comparing species, it appears that human microsomes, both of liver and
#
points (R
0.982, 0.971 and 0.898 for hydrolysis by human, rat and mouse lung microsomes
5
-
respectively). (c) C GE (1 dodecyl glycidyl ether). The lines represent model simulations for the
"#
Michaelis Menten equation Ž tted to the data points (R
#
0.992, 0.987 and 0.961 for hydrolysis
±
5
-
-
by human, rat and mouse lung microsomes respectively). (d) o CGE (o cresyl glycidyl ether).
The lines represent model simulations for the Michaelis Menten equation Ž tted to the data points
±
#
(R
0.821, 0.975 and 0.994 for hydrolysis by human, rat and mouse lung microsomes
5
-
respectively). (e) HDDGE (1,6 hexanediol diglycidyl ether). The lines represent model
simulations for the Michaelis Menten equation Ž tted to the data points (R
0.946 for hydrolysis by human, rat and mouse lung microsomes respectively).
#
0.978, 0.964 and
±
5

P J Boogaard
. .
496
et al.
-
Table 7. Apparent kinetic constants for enzymatic hydrolysis of 1 dodecanol glycidyl ether by liver and
lung cytosol and microsomes.
}
Vmax Km
ll min mg
V
Km
(mm)
max
}
}
}
}
Organ
Liver
Fraction
cytosol
Species
(nmol mg min)
n
R
human
rat
mouse
21.1
1.05
12.7
0.17
0.023
0.10
124
45.4
129
6
2
2
0.9995
0.999
0.969
microsomes
cytosol
human
rat
mouse
137
48.2
40.3
0.71
0.31
0.36
192
156
111
6
2
2
0.984
0.997
0.983
Lung
human
rat
mouse
0.77
0.21
0.22
0.20
0.78
0.076
3.88
0.26
2.89
6
2
2
0.950
0.953
0.997
microsomes
human
rat
mouse
10.9
6.0
11.7
0.076
0.069
0.072
143
87.3
163
6
2
2
0.996
0.993
0.983
}
n, number of individuals animals.
æ
R, correlation coe cient of the Ž tted curve.
-
Table 8. Apparent kinetic constantsfor enzymatichydrolysis of o cresylglycidyl ether by liver and lung
cytosol and microsomes.
}
Vmax Km
ll min mg
V
Km
(mm)
max
}
}
}
}
Organ
Liver
Fraction
cytosol
Species
(nmol mg min)
n
R
human
rat
mouse
12.7
1.16
45.8
0.062
0.019
1.60
204
60.2
28.6
6
2
2
0.981
0.995
0.9999
microsomes
cytosol
human
rat
mouse
106
61.4
23.2
0.43
0.26
0.086
244
232
268
6
2
2
0.966
0.984
0.989
Lung
human
rat
mouse
0.40
no reaction
0.91
0.013
±
3.04
30.7
±
0.30
6
4
3
0.985
±
0.998
microsomes
human
rat
mouse
6.86
2.68
8.08
0.036
0.032
0.049
189
84.5
165
6
2
2
0.906
0.987
0.997
}
n, number of individuals animals.
æ
R, correlation coe cient of the Ž tted curve.
}
V
max
K
m , towards
lung, have in general, a higher hydrolytic activity, as expressed by
the GE than rat or mouse microsomes (tables 5±9). The only exceptions being the
}
Vmax Km
o-
ratio for HDDGE in rat liver microsomes, for CGE in mouse liver
microsomes and for C GE in mouse lung microsomes. In these latter three cases,
"#
}
V
Km
ratio of the human microsomes roughly equalled those of the rodent
the
max
microsomes and had the lowest hydrolytic capacity towards the GE, the only
exception being the activity of liver microsomes towards HDDGE where rat and
}
Vmax Km
human are equal. The
ratio for hydrolysis was highest in human lung
}
V
K
cytosol for all GE except for YX4000 for which
m was higher in mouse lung
was higher in human liver cytosol than in mouse liver cytosol for
CGE and HDDGE, and the opposite was found for YX4000 and BADGE,
whereas about equal values were observed for C GE.
max
}
V
max
Km
cytosol.
o-
"#

Metabolic inactivation of glycidyl ethers
497
-
Table 9. Apparent kinetic constants for enzymatichydrolysis of 1,6 hexanediol diglycidyl ether by liver
and lung cytosol and microsomes.
}
Km
ll min mg
V
Km
(mm)
V
max
max
}
}
}
}
Organ
Liver
Fraction
cytosol
Species
(nmol mg min)
n
R
human
rat
mouse
36.6
3.48
96.9
1.05
1.01
3.07
34.7
3.45
31.5
6
2
3
0.992
0.989
0.9999
microsomes
cytosol
human
rat
mouse
93.7
23.0
18.9
0.77
0.18
0.26
122
129
74.0
6
2
2
0.999
0.984
0.977
Lung
human
rat
mouse
0.52
1.42
3.31
0.11
2.42
6.69
4.78
0.59
0.49
6
2
2
0.982
0.9991
0.9995
microsomes
human
rat
mouse
7.57
3.11
6.98
0.24
0.22
0.33
31.6
14.3
21.1
6
2
2
0.989
0.983
0.972
}
n, number of individuals animals.
æ
R, correlation coe cient of the Ž tted curve.
Discussion
The epoxy moiety makes glycidyl ethers chemically reactive compounds, a
characteristic which on the one hand is an essential prerequisite for their commercial
use, but on the other hand makes them toxicologically active since some epoxy
compounds have been associated with cancer. In a small number of cases, glycidyl
ethers have been reported to cause tumours in animal studies. For example, in an
inhalation study of phenyl glycidyl ether (PGE) in rat, nasal carcinomas, squamous
et
cell metaplasia, hyperplasia and dysplasia and chronic rhinitis were observed (Lee
al
. 1983). Based on this latter study, the IARC classiŽ ed PGE as a group 2B
carcinogen (possibly carcinogenic to humans) (IARC 1989, 1999a). Other glycidyl
ethers such as BADGE and triethylene glycol diglycidyl ether, were considered not
classiŽ able as to their carcinogenicity to humans (group 3) since only limited or
inadequate evidence in experimentalanimals and no epidemiologicaldata in humans
relevant to their carcinogenicity was available (IARC 1989, 1999c, d). The epoxy
group is also responsible for the direct mutagenic eåects observed in bacteria
et al
exposed to glycidyl ethers (Gardiner
. 1992)and for the DNA adducts observed
in vitro
et al
et al
. 1998).However,these observations
in
systems(Plna
. 1996,Deforce
in vivo
might be less relevant for the
situation since GE may be detoxiŽ ed by
hydrolysis of the epoxide group or by GSH conjugation. In the present study,
these two detoxiŽ cation reactions were quantiŽ ed for representatives of four
classes of GE.
Glutathione conjugation
All GE were chemically stable in an aqueous environment as no measurable rate
of hydrolysis was observed at maximum solubility. A small, but measurable,
-
o-
chemical reaction of GSH with the two most water soluble GE, CGE and
P
/
o w
HDDGE, with estimated log
2.2 and 0.84 respectively, was observed under
5
the given experimental conditions. Upon incubation with both liver and lung
o-
cytosol, rapid GSH conjugation was observed for both CGE and HDDGE(Ž gure
3). The observed conjugation rates were considerably lower in human cytosol than

P J Boogaard
. .
498
et al.
±
±
"
"
3
3
kg ) of various glycidyl ethers
Table 10. Estimated rate constantsfor in vivo hepatic clearance(l
h
in human and rodentsa.
Cytosolic glutathione
-
Glycidyl ether
YX4000
Species
S transferase
Epoxide hydrolase
human
rat
mouse
ndb
nd
51
45
103
!
LOQ
BADGE
human
rat
mouse
nd
LOQ
LOQ
17
13
35
!
!
C
GE
human
rat
mouse
nd
nd
LOQ
16
14
23
"#
!
-
o CGE
human
rat
mouse
0.45
3.6
3.7
24
19
10
HDDGE
human
rat
mouse
0.38
7.2
6.4
6.5
5.7
6.5
a See the Materials and methods for calculations.
b
!
nd, Not determined; LOQ: below limit of quantitation.
in rodent cytosol, which is in agreement with the relative rates of GSH conjugation
-
- -
-
observed for other epoxides such as 1,2 epoxy 3 butene and 1,2:3,4 diepoxybutane
et al et al
(Csanady
. 1992, Boogaard
. 1996, 1999).
o-
In contrast to CGE and HDDGE, only a very limited chemical reaction with
P /
o w
GSH was observed with BADGE (estimated log
3.8) and no chemical
5
P
/
o w
reaction with the most lipophilic GE C GE and YX4000 (estimated log
5.0
5
"#
and 5.1 respectively), which is probably explained by their poor solubility in water.
Low, inconsistent GSH conjugation with BADGE and no measurable GSH
conjugation with YX4000 and C GE were observed upon incubation with liver
"#
"%
et al
lm
-
C BADGE with
cytosol. Climie
. (1981b) reported that incubation of 6
}
m
CF1 mouse liver cytosol (2 mg protein ml) for 30 min in the presence of 5 m
GSH, gave a polar compound which did not migrate in a TLC system (ethyl
acetate±formic acid±water [70:4:4] on silicagel) and which was assumed to be the
GSH conjugate since the compound was not found in the absence of GSH.
-
Although this non migrating compoundwas not identiŽ ed, 80% of the radioactivity
bis-
applied to the TLC plate migrated and was recovered as the
diol of BADGE. In
-
-
our system, EH activity was inhibited by pre incubation with 4 PCO and only
bis-
traces of the diol and
diol could be identiŽ ed (data not shown). In the current
study, the formation of a GSH conjugate of BADGE was conŽ rmed in two ways.
-
First, small radio peaks with identical retentions appeared in both conjugation
$
-
experiments with H GSH and unlabelled BADGE and with unlabelled GSH and
"%
-
-
C BADGE. Second, the identity of the peak was conŽ rmed by LC MS.
-
In general, it seems that the more hydrophilic the GE is, the faster its enzyme
catalysed GSH conjugation proceeds. This is in agreement with the relative GSH
conjugation rates for substituted phenyl glycidyl ethers in Sprague±Dawley rat liver
}
}
p- -
-
cytosol: 17.7, 25.3 and 77.74 nmol mg protein min for methoxyphenyl , phenyl
p- et al
-
and nitrophenyl glycidyl ether (Sinsheimer . 1987), which are closely related
}
}
o-
to CGE for which a
V
max
61.3 nmol mg protein min was measured in rat liver
5
cytosol in our study. Although little, if any, GSH conjugation was measured for

Metabolic inactivation of glycidyl ethers
499
±
±
"
"
3
3
kg ) of various glycidyl
Table 11. Estimated rate constants for in vivo pulmonary clearance (l
h
ethers in human and rodentsa.
Cytosolic glutathione
-
Glycidyl ether
YX4000
Species
S transferase
Epoxide hydrolase
human
rat
ndb
nd
1.9
0.87
mouse
nd
2.6
BADGE
human
rat
mouse
nd
nd
nd
0.28
0.13
0.17
C
GE
human
rat
mouse
nd
nd
nd
0.17
0.066
0.33
"#
-
o CGE
human
rat
mouse
0.02
0.07
0.03
0.40
0.060
0.32
HDDGE
human
rat
mouse
0.01
0.22
0.05
0.064
0.018
0.043
a
See the Materials and Methods and Materials for calculations.
nd, Not determined.
b
YX4000, BADGE and C GE, it is clear from the estimated values in tables 10 and
"#
o-
11 that GSH conjugation is an important route of detoxiŽ cation for CGE and
HDDGE,especially in rodents. The estimated hepatic clearances were 0.45, 3.6 and
} }
o-
for HDDGE in human, rat and mouse respectively. In a identical way,
3.7 l h kg for CGE in human, rat and mouse respectively, and 0.38, 7.2 and 6.4
in vivo
in vitro
clearances were estimated based on
data for a limited number of simple
aliphatic epoxides. Comparedwith these epoxides, the estimated clearances by GSH
} }
conjugationare strikingly similar: 0.47, 5.66 and 4.36 l h kg for hepatic clearance of
- - et al
-
1,2 epoxy 3 butene by human, rat and mouse respectively (Csanady . 1993)and
} }
-
0.26, 2.51 and 7.82 l h kg for hepatic clearance of 1,2:3,4 diepoxybutane by
et al
. 1996,Boogaardand Bond 1996).
human, rat and mouse respectively (Boogaard
-
o-
This indicates that, with regard to enzyme catalysed GSH conjugation, CGE and
HDDGE behave like simple aliphatic epoxides.
Epoxide hydrolysis
Epoxide hydrolysis is the most important route of detoxiŽ cation for most GE.
bis-
All GE were rapidly hydrolysed to the corresponding (
)diols. The two glycidyl
-
groups in the bifunctional GE, YX4000, BADGE and HDDGE, behave in
dependently. This is in agreement with the results for BADGE reported by Climie
et al
. (1981a). Apparently, the aænity of the EH for the unchanged GE and the
-
corresponding mono diol is equal or only negligibly diåerent, which allows the use
-
bis-
of the total of the mono diol and
diol to calculate the Michaelis±Menten
parameters. There is a considerable diåerence between the various GE in eæciency
}
V
max
K
m . In general, the microsomalEH is more eæcient
of hydrolysis, expressed as
than the cytosolic EH, and human microsomal fractions are more eæcient in
-
hydrolysis of the GE than rodent microsomal fractions at low substrate concen
trations, which are more relevant for human exposure. Although no simple
relationship was found, the reaction catalysed by the microsomal EH seems to be

P J Boogaard
. .
500
et al.
related with the aromaticity and lipophilicity of the compounds. GE with an
}
K
}
K
m is higher for GE
V
max
V
max
aromaticring have higher
m than aliphatic GE, and
}
P
Vmax Km
with a higher log o w . For human liver microsomes, for instance, the
/
"
"
"
"
o-
decrease in the following order YX4000 BADGE
in vivo
CGE C GE
clearance for the Ž ve GE (tables 10 and 11)indicate
"#
HDDGE.The estimated
that rat has the poorest clearance, both in liver and lung, with exception of the
o-
hepatic clearance of CGE, which is least in mouse. Generally, there is not much
diåerence in hepatic clearance: it diåers no more than 1 order of magnitude from
one GE to the other and from one species to the other. The diåerences in pulmonary
!
clearance are larger: although there is 1 order of magnitude of diåerence between
species for the same GE, much larger diåerences are found between the various GE.
in vivo
Despite this variation, on the whole the
much larger (about 2 orders of magnitude) than for simple aliphatic epoxides such
- - et al
clearances for both liver and lung are
-
-
as 1,2 epoxy 3 butene and 1,2:3,4 diepoxybutane (Csanady . 1993, Boogaard
and Bond 1996). In experimental animals, the relative importance of epoxide
in vivo
hydrolysis compared with GSH conjugation was demonstrated
for PGE and
CGE. Upon administration of PGE to rat and rabbit, either by oral gavage or by
- -
o-
-
-
subcutaneous injection, the major urinary metabolite was 2 hydroxy 3 phenoxy
proprionic acid, formed by hydrolysis of the epoxy group of PGE and subsequent
N- -S- - - -l-
-
oxidation. In addition,
acetyl (2 hydroxy 3 phenoxypropyl) cysteine, the
mercapturic acid of PGE formed following GSH conjugation of PGE, was excreted
et al
as a minor metabolite in urine (James
. 1978). More recently, the urinary
metabolite proŽ le of PGE and CGE was studied in rat following intraperitoneal
et al
o-
administration (De Rooij
. 1998). In this latter study a novel metabolite of PGE
phenylserine, which is believed to result from oxidation of
N-
N- -O-
was found:
- -
acetyl
-
-
acetyl
2 hydroxy 3 phenoxyproprionic acid followed by transamination and
ation, and thus a metabolite from the epoxide hydrolysis pathway. A similar
o-
metabolite was also found for CGE. At low doses of PGE the GSH pathway
"
accountedfor 25% of the metabolites excreted in urine, and with increasing dose,
C
this percentage declined to
10 %. In contrast, the epoxide hydrolysis pathway
o-
C
accounted for 40% of the urinary metabolites, irrespective the dose. For CGE
et al
. 1998).
a similar pattern was observed (De Rooij
Oxidative dealkylation
Theoretically, metabolism of GE might also lead to more toxic compounds since
-
oxidative de alkylation of the GE might lead to the formation of glyceraldehyde and
glycidaldehyde. The latter is classiŽ ed by IARC as a group 2B carcinogen (possibly
carcinogenic to humans) (IARC 1976, 1999e). Although it has been argued that
in vivo
et al
(Climie . 1981a, b),
glyceraldehyde, and not glycidaldehyde, is formed
there is still concern about the formation of glycidaldehyde as its formation from
in vitro et al
PGE and allyl glycidyl ether has been demonstrated
(Plna
. 1996.
et al in vivo
Deforce
. 1998). Evidence for the
BADGE and allyl glycidyl ether has also been reported following topical adminis
et al et al
formation of glycidaldehyde from
-
tration (Bentley
. 1989, Steiner . 1992a, b, Plna and Segerback 1997). It has
-
previously been shown that oxidative de alkylation of BADGE only occurred at
high doses, leading to saturation of the EH pathway, or when EH was inhibited
et al
(Bentley . 1989). Like BADGE, glycidaldehyde is a good substrate for EH and
glycidaldehyde is a particularly good substrate for GST. GSH conjugation rates in

Metabolic inactivation of glycidyl ethers
501
m
m
rat, after incubation of 3.0 m glycidaldehyde with 3.0 m GSH, were 4.1 0.4 and
6
}
}
}
}
l
1.9 0.1 mol mg protein min for liver (1 mg ml) and lung (1.5 mg ml) cytosol
6
m
respectively, and hydrolysis rates, after incubation of 0.1 m glycidaldehyde with
}
}
}
}
liver (3.3 mg ml) or lung (5 mg ml) microsomes, were 26 and 20 nmol mg min
et al
respectively (Patel
. 1980). Accordingly, it is unlikely that the formation of
glycidaldehyde from BADGE or other GE will pose an important genotoxic risk as
the small amounts of glycidaldehyde will be rapidly detoxiŽ ed.
In conclusion, the selected GE, which represent diåerent classes of GE, show a
very large variation with regard to the rate of inactivation of the epoxide moiety
indicating that GE have highly variable properties and should not be considered as
a single class of compounds. In addition, the rates for hydrolysis of the epoxide
C
moiety, the most important inactivation pathway for most of the GE, are 2 orders
of magnitude higher than for some simple aliphatic epoxides.
References
-
Bentley , P., Bieri , F., Kuster, H., Muakkassah Kelly , S., Sagelsdorff , P., Staubli , W. and
Waechter , F., 1989,Hydrolysis of bisphenol A diglycidylether by epoxide hydrolases in cytosolic
and microsomalfractionsof mouse liver and skin: inhibition by bis epoxycyclopentylether and the
å
e ects upon the covalent binding to mouse skin DNA. Carcinogenesis, 10, 321 327.
±
-
Boogaard, P. J. and Bond, J. A., 1996, The role of hydrolysis in the detoxiŽ cation of 1,2:3,4
diepoxybutane by human, rat, and mouse liver and lung in vitro. Toxicology and Applied
Pharmacology, 141, 617 627.
±
Boogaard, P. J., Denneman , M. A. and van Sittert , N. J., 2000, Dermal penetration and metabolism
of Ž ve glycidyl ethers in human, rat and mouse skin. Xenobiotica, 30, 469 483.
Boogaard, P. J., Sumner , S. C. J. and Bond, J. A., 1996, Glutathione conjugation of 1,2:3,4
±
-
diepoxybutane in human liver and rat and mouse liver and lung in vitro. Toxicology and Applied
Pharmacology, 136, 307 316.
±
Boogaard, P. J., van Elburg, P. A., de Kloe, K. P., Watson , W. P. and van Sittert , N. J., 1999,
-
-
-
-
Metabolic inactivation of 2 oxiranomethyl 2 ethyl 2,5 dimethyl hexanoate (C GE) in skin,
"!
lungs, and liver of humans, rats and mice. Xenobiotica, 29, 987 1006.
±
-
Climie , I. J. G., Hutson , D. H. and Stoydin , G., 1981a,Metabolism of the epoxy resin component 2,3
-
-
bis[4 (2,3 epoxypropoxy)phenyl]propane, the diglycidyl ether of bisphenol A (DGEBPA) in the
"%
-
mouse. I. A comparison of the fate of a single dermal application and of a single oral dose of
DGEBPA. Xenobiotica, 11, 391 399.
Climie , I. J. G., Hutson , D. H. and Stoydin , G., 1981b, Metabolism of the epoxy resin component 2,3
C
±
-
-
-
bis[4 (2,3 epoxypropoxy)phenyl]propane, the diglycidyl ether of bisphenol A (DGEBPA) in the
"%
-
C
mouse. II. IdentiŽ cation of metabolites in urine and faeces following of a single oral dose of
DGEBPA. Xenobiotica, 11, 401 424.
±
Csanady, G. A., Guengerich , F. P. and Bond, J. A., 1992a, Comparison of the biotransformation of
-
1,3 butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from
humans, rats and mice. Carcinogenesis, 13, 1143 1153. [Erratum Carcinogenesis, 14, 784.]
±
De Rooij , B. M., Commandeur , J. N. M., Hommes , J. W., Aalbers , T., Groot, E. J. and Vermeulen ,
-
N. P. E., 1998, Urinary metabolite proŽ le of phenyl and o cresyl glycidyl ethers in rats:
-
-
identiŽ cation of a novel pathway leading to N acetylserine O conjugates. Chemical Research in
Toxicology, 11, 111 118.
±
Deforce , D. L. D., Lemie re, F., Hoes, I., Millecamps , R. E. M., Esmans , E. L., de Leenheer , A. and
van den Eeckhout , E. G., 1998, Analysis of the DNA adducts of phenyl glycidyl ether in a calf
thymus DNA hydrolysate by capillary zone electrophoresis electrospray mass spectrometry:
±
evidence for phosphate alkylation. Carcinogenesis, 19, 1077 1086.
±
}
-
æ
Eadsforth , C. V., 1983, EPIKOTE 828: Determination of the N Octanol water Partition Coe cient
-
Using a Reverse phase HPLC Method. Report no. SBGR 83. 385 (Sittingbourne : Shell Research
Centre).
Gardiner , T. H., Waechter , J. M., Wiedow , M. A. and Solomon , W. T., 1992, Glycidyloxy
compounds used in epoxy resin systems: a toxicology review. Reviews in Toxicology and
Pharmacology, 15, S1 77.
Guest, D. and Dent , J. G., 1980, E ects of epoxide hydratase inhibitors in forward and reversion
±
å
bacterial mutagenesis assay systems. Environmental Mutagenesis, 2, 27 34.
±
IARC, 1976, Cadmium, Nickel, some Epoxides, Miscellaneous Industrial Chemicals and General
Considerations on Volatile Anaesthetics. Glycidaldehyde (Lyon: IARC), 11, 175 181.
±

Metabolic inactivation of glycidyl ethers
502
IARC, 1985, Allyl Compounds, Aldehydes, Epoxides and Peroxides. Diglycidyl Resorcinol Ether (Lyon:
IARC), 36, 181 188.
±
IARC, 1989,Some Organic Solvents, Resin Monomersand Related Compounds, Pigments and Occupational
Exposures in Paint Manufactureand Painting. Some Glycidyl Ethers (Lyon: IARC), 47, 237 261.
±
-
IARC, 1999a, Re evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. Phenyl
Glycidyl Ether (Lyon: IARC), 71, 1525.
-
IARC, 1999b, Re evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. Diglycidyl
Resorcinol Ether (Lyon: IARC), 71, 1417.
-
IARC, 1999c, Re evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. Bisphenol A
Diglycidyl Ether (Lyon: IARC), 71, 1265.
-
IARC, 1999d, Re evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. Triethylene
Glycol Diglycidyl Ether (Lyon: IARC), 71, 1285.
-
IARC, 1999e, Re evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide.
Glycidaldehyde (Lyon: IARC), 71, 1459.
ILSI, 1994, PhysiologicalParameterValuesfor PBPK Models. Report prepared by the ILSI Risk Science
Institute Working Group on Physiological Parameters (Washington, DC: International Life
Science Institute).
-
- - -
-
James , S. P., Pheasant , A. E. and Solheim , E., 1978, Metabolites of 1,2 epoxy 3 phenoxy and 1,2
- -
-
epoxy 3 (p nitrophenoxy)propane. Xenobiotica, 8, 219 228.
±
Lee, K. P., Schneider , P. W. and Trochimowicz , H. J., 1983, Morphological expression of glandular
å
di erentiation in the epidermoid nasal carcinomas induced by phenyl glycidyl ether inhalation.
American Journal of Pathology, 3, 140 148.
±
Mullin , C. A. and Hammock , B. D., 1982, Chalcone oxides potent selective inhibitors of cytosolic
±
epoxide hydrolase. Archives of Biochemistry and Biophysics, 216, 423 439.
±
Patel, J. M., Wood, J. C. and Leibman , K. C., 1980,The biotransformationof allyl alcohol and acrolein
in rat liver and lung preparations. Drug Metabolism and Disposition, 8, 305 308.
±
$#
-
Plna, K. and Segerback, D., 1997, P postlabelling of DNA adducts formed by allyl glycidyl ether in
vitro and in vivo. Carcinogenesis, 18, 1457 1462.
±
Plna, K., Segerback, D. and Schweda, E. K. H., 1996, DNA adduct formation by allyl glycidyl ether.
Carcinogenesis, 17, 1465 1471.
±
Sinsheimer , J. E., van den Eeckhout , E., Hooberman , H. B. and Beylin , V. G., 1987, DetoxiŽ cation
-
of aliphatic epoxides by diol formation and glutathione conjugation. Chemico Biological
Interactions, 63, 75 90.
±
Steiner , S., Crane, A. E. and Watson , W. P., 1992a, Molecular dosimetry of DNA adducts in C3H
mice treated with glycidaldehyde. Carcinogenesis, 13, 119 124.
±
Steiner , S., Honger, G. and Sagelsdorff , P., 1992b, Molecular dosimetry of DNA adducts in C3H
mice treated with bisphenol A diglycidyl ether. Carcinogenesis, 13, 969 972.
Van Elburg, P. A., Ormskerk , O. A., de Kloe, K. P. and Boogaard, P. J., 2000, Synthesis of [ C]
±
"%
-
labelled glycidyl and glycerol ethers of aliphatic and aromatic alcohols. Journal of Labelled
Compound Radiopharmacology, 43, 147 167.
±