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EC number: 203-794-9
CAS number: 110-71-4
NADPH-dependent metabolism of Diglyme by rat hepatic microsomes
Diglyme metabolites were determined using radiolabelled Diglyme and HPLC
analysis. Three major metabolites were detected in rat hepatic
microsomal incubations containing an NADPH-generating system. Two of the
metabolites were identified as 2 -Methoxyethanol and 2 -(2
-Methoxyethoxy)ethanol. The identity of the third metabolite was not
determined.Analysis of incubations containing rat hepatic microsomes
from untreated rats, an NADPH-generting system and 1 mM Diglyme indicate
less than 2% of the added Diglyme was metabolized after 30 min.
Metabolism of Diglyme by human hepaic microsomes
Human hepatic microsomes obtained from eight subjects were assayed for
Aniline hydroxylase activity. The mean value for the amount of Aniline
metabolized/min/nmol P-450 for humans was 0.60. Haevy alcohol use was
reported in the case histories of two of the subjectes. the Aniline
hydroxylase activities measured in microsomes isolated from these
subjects were equal to or greatr than the mean but were not
substantially elevated over the other measured values.
Human hepatic microsomes catalysed the NADPH-dependent cleavage of
Diglyme to 2 -Methoxyethanol. In addition to 2 -Methoxyethanol, 2 -(2
-Methoxyethoxy)ethanol and an unknown metabolite were also identitfied
in human microsomal incubations containing radiolabelled Diglyme.
Formation of these products was demonstrated to be NADPH-dependent.
These productes were also identified with rat hepatic microsomes
containing an NADPH-generating system. A mena value of 19.7 nmol 2
-Methoxyethanol/min/nmol P-450 was determined for the 30 min incubations
with human microsomes.
A test of the association between the Aniline hyroxylase activity
(nmol/min/mg protein) of human microsomes and the amount of 2
-Methoxyethanol (nmol/mg protein) formed by these microsomes during a 30
min incubation yielded a correlation coefficient of 0.87. In a similar
manner, the combined Aniline hydroxylase and the 2 -Methoyethanol
results from untreated and Ethanol-, Phenobarbital-, diglyme- and
ß-Naphthoflavone-preteated rats yielded a correlation coefficient of
0.71. The association between these two viariables for human and rat
hepatic microsomes was statistically significant (p<0.01).
Effects of P-450 induces on 2 -Methoxyethanol formation (rat
Treatment of rats with drinking water containing Diglyme before they
were killed significantly increased microsomal levles of P-450 (p<0.05).
P-450 levels increased by 70% above control values. Diglyme pretreatment
also significantly increased P-450 -associated enzyme activities.
Statisitcally significant increases were noted in the specific activity
levels of Ethoxycoumarin deethylase and pentoxyresorufin dealkylase
following Diglyme pretreatment. Pretreament of animals with either
Ethanol or Phenobarbital augmented the metabolism of Diglyme to 2
-Methoxyethanol catalysed by rat hepatic microsomes. Both the ethanol-
and phenobarbital-induced increases were determined significant
regardless of whether the amount of 2 -Methoxyethanol formed was
expressed per mg protein or per nmol P-450. Pretreatment of animals with
Diglyme also significantly (p<0.05) increased the capacity of microsomes
to convert Diglyme to 2 -Methoxyethanol.
Time- and protein-dependent formation of 2 -Methoxyethanol (rat
The effects of incubation time on the cleavage of Diglyme to 2
-Methoxyethanol by phenobarbital-induced rat hepatic microsomes were
examined. 2 -Methoxyethanol formation was time dependent, and measured
rates of formation for 2 -Methoxyethanol were similar over the first 30
min of the experiment (approx. 0.5 nmol/min). After 30 min, rates of 2
-Methoxyethanol formation appeared to diminish slightly with increasing
incubation time. The amount of Diglyme cleaved to 2 -Methoxyethanol
during 30 min incubations with phenobarbital-induced microsomes was also
dependent on the concentration of microsomal protein present in the
incunation media. Increasing concentrations of microsomal protein
produced proportionally greater amounts of 2 -Methoxyethanol up to about
1 mg protein/mL. Additions of microsomal protein above 1.5 mg/mL did not
increase the amount of 2 -Methoxyethanol formed during 30 min
Effects of P-450 inhibitor on 2 -Methoxyethanol formation
Metyrapone (inhibitor of phenobarbital-induced P-450), TAO (specific
inhibitor of P450 III) and Isoniazid (inhibitor of P450 IIIEI)
significantly reduced the cleavage of Diglyme to 2 -Mthoxyethanol
catalysed by phenobarbital-induced microsomes. Isiniazid produced a 67 %
inhibition of 2 -Methoxyethanol formation in phenobarbital-induced
microsomes, while Metyrapone and TAO produced inhibitions of 34 and 51
%, respectively. Only Isoniazid significantly reduced the cleavage of
Diglyme to 2 -Methoxyethanol catalysed by microsomes isolated from
ethanol- or diglyme-pretreated rats. Isoniazid inhibited 2
-Methoxyethanol formation by 85 % with ethanol-induced microsomes and by
53 % with microsomes isolated from diglyme-pretreated rats.
Due to the high structural similarity of Ethylene glycol dimethyl ether
and Diethylene glycol dimethyl ether (difference: one ethyl group; but
same functional groups) it is very likely that both compounds will be
metabolised by the same enzymes/metabolic path. Therefore, a read across
from Ethylene glycol dimethyl ether to the metabolism data generated
with Diethylene glycol dimethyl ether (Diglyme) is jusitfied to clarify
the toxicokinetic behaviour of this substance (for further clarification
please refer to the attached document "Proposed metabolism of Ethylene
glycol dimethyl ether.doc").
Human and rat hepatic microsomes were incubated with Diethylene glycol
dimethyl ether (Diglyme). The rat microsomes catalysed the
NADPH-dependent cleavage of the central ether linkage of Diglyme
yielding 2 -Methoxyethanol and 2 -(2 -Methoxyethoxy)ethanol. Microsomes
isolated from phenobarbital- or ethanol-pretreated rats exhibited an
increased capacity to cleave diglyme to 2 -Methoxyethanol. This
ethanol-induced increase in 2 -Methoxyethanol formation was not observed
if incubations contained the cytochrome P450 IIEI inhibitor Isoniazid.
Pretreatment of rats with Diglyme significantly increased microsomal
P-450 levels, P-450 associated enzyme activities and the conversion of
Diglyme to 2 -Methoxyethanol.
Human hepatic microsomes also catalysed the NADPH-dependent cleavage of
Diglyme to 2 -Methoxyethanol. The formation of 2 -Methoxyethanol from
Diglyme correlated with the aniline hydroxylase activity (P450 IIEI)
levels measured in human hepatic microsomes.
These results suggest that the (central) ether linkage of Diglyme (and
Ethylene glycol dimethyl ether) is cleaved by rat and human P-450 and 2
-Methoxyethanol is formed.
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