In vivo and in vitro formation of morphinone from morphine in rat

Article abstract of DOI:10.1080/004982597240244

1. Morphinone, a toxic metabolite, and its glutathione adduct (MO-GSH) were identified in the bile of rat after subcutaneous injection of morphine (25 mg/kg) by hplc procedures. The amounts of morphinone and MO-GSH excreted in the 12-h bile were 0.8 ± 0 3 and 8.4 ± 4.3% respectively. 2. The 9000 g supernatants of rat, guinea pig, rabbit, mouse, hamster and bovine livers produced morphinone from morphine in the presence of either NAD⁺ or NADP⁺. NAD⁺ was a more efficient cofactor than NADP⁺ except in the guinea pig which equally utilized both cofactors. With NAD⁺ as cofactor, the amounts of morphinone formed in rat and guinea pig were 5.70 and 5.82 μmol/g liver/30 min respectively and were three-to-four times those in other species. 3. The enzyme activity responsible for formation of morphinone from morphine in the rat was almost exclusively distributed in the microsomal fraction, whereas guinea pig, hamster and bovine expressed the enzyme activity mainly in the cytosolic fraction. Rabbit and mouse gave higher activity in the cytosolic and microsomal fractions respectively, but other fractions of both species contained considerable activity. 4. The enzyme activities in male and female rat microsomes were characterized with respect to developmental pattern, kinetic parameters, pH dependency and susceptibility to inhibitors. 5. In conclusion the metabolism of morphine to morphinone in rat was confirmed by in vivo and in vitro experiments. It is also suggested that this pathway is a common route in morphine metabolism in several mammalian species.

Full text of DOI:10.1080/004982597240244

xenobiotica , 1997, vol. 27, no. 7, 645±656
and
form ation of m orphinone from
in vitro
In vivo
m orphine in rat
S. YAMANO*, A. TAKAHASHI, T. TODAKA and S. TOKI
Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma 8-19-1, Jonan-ku,
Fukuoka 814-80, Japan
Received 10 January 1997
1. Morphinone, a toxic metabolite, and its glutathione adduct (MO-GSH) were
identi®ed in the bile of rat after subcutaneous injection of morphine (25 mg kg) by hplc
}
procedures. The amounts of morphinone and MO-GSH excreted in the 12-h bile were
±
±
±
±
0 8³0 3 and 8 4³4 3% respectively.
2. The 9000 g supernatants of rat, guinea pig, rabbit, mouse, hamster and bovine livers
+
+
produced morphinone from morphine in the presence of either NAD or NADP . NAD
+
+
was a more eæcient cofactor than NADP except in the guinea pig which equally utilized
+
both cofactors. With NAD as cofactor, the amounts of morphinone formed in rat and
±
guinea pig were 5 70 and 5 82 mol g liver 30 min respectively and were three-to-four
±
l
}
}
times those in other species.
3. The enzyme activity responsible for formation of morphinone from morphine in the
rat was almost exclusively distributed in the microsomal fraction, whereas guinea pig,
hamster and bovine expressed the enzyme activity mainly in the cytosolic fraction. Rabbit
and mouse gave higher activity in the cytosolic and microsomal fractions respectively, but
other fractions of both species contained considerable activity.
4. The enzyme activities in male and female rat microsomes were characterized with
respect to developmental pattern, kinetic parameters, pH dependency and susceptibility to
inhibitors.
5. In conclusion the metabolism of morphine to morphinone in rat was con®rmed by
in vivo and in vitro experiments. It is also suggested that this pathway is a common route
in morphine metabolism in several mammalian species.
Introduction
Morphinone, identi®ed in the urine (Ishida et al. 1981) and the bile (Kumagai et
al. 1987) of guinea pigs given morphine, is nine times more toxic than morphine
(Nagamatsu et al. 1982a) and is a potent antagonistic of morphine (Nagamatsu et al.
1982b) in mouse. This metabolite was also found to block irreversibly naloxone
binding in the mitochondrial-synaptosomal fraction of mouse brain (Nagamatsu et
al. 1982b), to bind covalently to tissue macromolecules through sulphhydryl groups
in mouse (Nagamatsu et al. 1983), and to cause hepatotoxicity in rat (Nagamatsu et
al. 1986, Nagamatsu and Hasegawa 1992). These ®ndings suggest that morphinone
formed from morphine may aåect morphine analgesia and has a potential role in
morphine toxicity including appearance of tolerance, although much of this
metabolite would be trapped by intracellular glutathione to form the morphinone±
glutathione adduct (MO-GSH) and excreted readily into the bile as demonstrated in
the guinea pig (Kumagai et al. 1990). It is therefore of interest and important to
assess the in vivo and in vitro formation of morphinone including MO-GSH in
various animals and man.
In the guinea pig, the formation of both morphinone and MO-GSH has been
* Author for correspondence.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

646
S. Yamano et al.
Figure 1. Morphinone formation and conjugation.
de®nitively demonstrated by in vivo and in vitro experiments (Kumagai et al. 1990),
as shown in ®gure 1. Morphine 6-dehydrogenase (EC 1\1\1\218) catalysing the
formation of morphinone from morphine in the liver of guinea pig has been puri®ed
±
a nd extensively characterized (Yamano et al. 1985, 1986). Morphinone, having an
a,b-unsaturated ketone group, non-enzymatically reacts with glutathione to form
MO-GSH by a Michael addition under physiological conditions (Nagamatsu et al.
1982a) and glutathione S-transferase also participates in this reaction (Kumagai et
al. 1990). MO-GSH isolated from the bile of guinea pig given morphine was
identi®ed as (8S)-(glutathione-S-yl)dihydromorphinone (Ishida et al. 1989). In the
rat, the formation of morphinone and MO-GSH has been reported in isolated
hepatocytes (Nagamatsu et al. 1986). In the mouse, the formation of MO-GSH has
been postulated after incubation of cytosolic fractions with morphine in the presence
+
of NAD and glutathione (Nagamatsu et al. 1983). However, except in the guinea
pig, the in vivo formation of morphinone and MO-GSH and the assessment of
morphine 6-dehydrogenase activity in the livers of various species have not been
reported.
In the current study, we describe the biliary excretion of morphinone and MO-
GSH in the rat after subcutaneous (s.c.) administration of morphine, and compare
the morphine 6-dehydrogenase activity in the livers of rat, guinea pig, mouse, rabbit
hamster and bovine. Some properties of rat microsomal enzyme activity are also
described.
M aterials and m ethods
Materials
Morphine hydrochloride was obtained from Takeda Chemical Ind., Ltd (Osaka, Japan), pyridine
nucleotides from Oriental Yeast Co., Ltd (Osaka, Japan), and steroids, naloxone hydrochloride and
indomethacin from Sigma Chemical Co. (St Louis, MO, USA). Morphinone was synthesized by the
method of Rapoport et al. (1957). Morphinone-mercaptoethanol adduct (MO-ME) and MO-GSH were
synthesized as described previously (Yamano et al. 1985, Ishida et al. 1989). The purity of these three
"
synthetic compounds ( 99 %) were checked by hplc. All other chemicals used were of the highest grade
available.

Morphinone formation in the rat
647
Animals
Male and female Wistar rats (3±12-weeks old, 40±400 g), male Hartley guinea pigs (400±500 g), male
±
Japanese white rabbits (2±2 5 kg), male, ddY mice (10±15 g) and male golden hamsters (100±150 g) were
fasted for 24 h before use, with water provided ad libitum. Bovine livers were obtained from a local
slaughterhouse.
Determination of morphinone and MO-GSH in rat bile
Adult male Wistar rats (350±400 g) were anesthesized with diethyl ether and the bile ducts cannulated
with polyethylene PE-10 tubing. Morphine (25 mg kg) was injected subcutaneously after 1 h of surgery
and the bile was collected every 1 h for 4 h and thereafter every 2 h for 8 h. The bile collected for 1 h
}
±
before administration of morphine was used as a control. Each bile sample was adjusted to 2 5 ml with
water, and divided into two portions to determine morphinone and MO-GSH formation separately. For
±
morphinone analysis, one aliquot (1 ml) was added to 0 2 sodium phosphate buåer, pH 7 4, containing
10 m 2-mercaptoethanol to convert morphinone to the morphinone 2-mercaptoethanol adduct (MO-
±
m
m
ME). The reaction mixture was poured onto a Sep-Pak C cartridge (Waters Division of Millipore Co.,
Milford, MA, USA). The cartridge was washed with 10 ml water and then eluted with 2 ml methanol.
")
The eluate was used for quanti®cation of morphinone by reversed-phase hplc on a column of YMC-Pak
m
ODS-ALL (150¬6 mm i.d., YMC Co., Kyoto, Japan) using a mobile phase consisted of 10 m sodium
±
±
phosphate buåer, pH 7 2 acetonitrile methanol (50 :45 :5 by vol.) at a ¯ow rate of 1 2 ml min. The hplc
}
}
}
apparatus consisted of a L-6200 Intelligent Pump, a L-4200 UV-VIS Detector and a AS-2000
Autosampler from Hitachi, Ltd (Tokyo, Japan). The peak area was determined on a Hitachi D-2500
Chromato-Integrator. The peaks were monitored for absorbance at 214 nm. Under these conditions, the
±
retention time of MO-ME was 6 5 min. The mean recovery of morphinoe from bile was 93 % (n ¯ 3).
±
The other aliquot of bile (1 ml), for MO-GSH analysis, was added to 0 2 sodium phosphate buåer,
m
±
pH 7 4, and poured onto a Sep-Pak C . The cartridge was washed with 1 ml water and then eluted with
")
3 ml 10 m sodium phosphate buåer, pH 3 15 acetonitrile (3: 1, v v). The eluate was subjected to hplc
±
m
}
}
on a Nova-Pak C cartridge (100¬8 mm i.d., Waters Division of Millipore Co.) using a mobile phase
")
consisted of 10 m sodium phosphate buåer, pH 3 15, containing 1 m sodium lauryl sulphate
±
m
m
}
±
acetonitorile (3 :1, v v) at a ¯ow rate of 1 2 ml min. The peaks were monitored for absorbance at 214 nm.
Under these conditions, the retention time of MO-GSH was 12 0 min. The mean recovery of MO-GSH
}
}
±
was 96 % (n ¯ 3).
In vitro formation of morphinone from morphine in rat, guinea pig, rabbit, mouse, hamster and bovine
livers
±
±
m
Livers were homogenized in 2 vols 0 1 sodium phosphate buåer, pH 7 4, with Poritron homogenizer
(Kinematika GmbH, Luzen, Switzerland). The homogenate was centrifuged at 9000 g for 20 min and the
resulting supernatant was further centrifuged at 105000 g for 60 min to sediment microsomes and a
±
cytosolic fraction. The microsomal pellet was washed twice with 1 15% KCl. Each fraction was adjusted
±
to a concentration of 0 33 g wet liver weight per ml with 0 1 sodium phosphate buåer, pH 7 4. The
incubation mixture consisted of morphine (3 3 m ), NAD or NADP (8 3 m ), enzyme fraction
±
±
m
+
+
±
±
m
m
±
equivalent to 0 165 g wet liver) and 0 1 sodium phosphate buåer, pH 7 4, in a total volume of 3 ml and
was incubated at 37 C for 30 min. Just before the reaction was terminated by immersing in a boiling
±
±
m
°
l
water bath for 2 min, 30 mol 2-mercaptoethanol was added to convert morphinone formed to MO-ME.
The reaction mixture was treated with Sep-Pak C , and morphine was quantitated by hplc as described
")
above.
Results
Biliary excretion of morphinone and MO-GSH in rat
Figure 2 shows a typical hplc pro®le of the bile extract obtained for morphinone
±
analysis. After morphine administration, a peak at retention time of 6 5 min
corresponding to authentic MO-ME was identi®ed (®gure 2B). This peak was not
detected in the control bile (®gure 2A). In this experiment, morphinone was
converted to MO-ME by adding 2-mercaptoethanol to the bile before extraction as
described in the Materials and Methods. When the extraction was carried out
±
without addition of 2-mercaptoethanol, the peak with retention time of 6 5 min
±
disappeared but a new peak with retention time of 7 5 min corresponding to
morphinone emerged and the extract obtained after addition of glutathione instead

648
S. Yamano et al.
±
Figure 2. Identi®cation of morphinone in bile. A portion of bile was added to 0 2 sodium phosphate
m
±
buåer, pH 7 4, containing 10 m 2-mercaptoethanol to convert morphinone to morphinone 2-
mercaptoethanol adduct (MO-ME) and then poured onto a Sep-Pak C cartridge. The extraction
m
")
procedure and hplc conditions are described in the Materials and methods. (A) Control rat bile;
(B) bile from rat given morphine (s.c. 25 mg kg). Arrow indicates the retention time of MO-ME.
}
Figure 3. Identi®cation of a glutathione conjugate of morphinone in bile. The extraction procedure and
hplc conditions are described in the Materials and methods. (A) Control rat bile ; (B) bile from rat
administered morphine (s.c. 25 mg kg). Arrow indicates the retention time of MO-GSH.
}
of 2-mercaptoethanol to the bile did not contain either peaks (data not shown).
±
Thus, these ®ndings indicate that the product with retention time of 6 5 min in
®gure 2B is MO-ME derived from morphinone.
Hplc chromatograms of the bile extract for MO-GSH analysis revealed a peak
±
with retention time of 12 0 min corresponding to MO-GSH (®gure 3B), which was
not present in the control bile (®gure 3A). Under the hplc conditions used, MO-
GSH has been shown to be separated from morphine 3-glucuronide (M-3-G),
±
which appeared at a retention time of 5 5 min as a large peak in ®gure 3B, morphine
6-glucuronide (M-6-G), morphine and other unconjugated metabolites (Kumagai
et al. 1990). However, Correia et al. (1984a) isolated 10a-S-glutathionylmorphine
after the incubation of morphine with liver microsomes prepared from the
phenobarbital-treated rat in the presence of glutathione and NADPH and this

Morphinone formation in the rat
649
Figure 4. Cumulative biliary excretion of morphinone and MO-GSH in the adult male rat. A single
dose of morphine (25 mg kg) was administered s.c. to seven bile duct-cannulated rats.
}
Morphinone (D ) and MO-GSH (E ) excreted in the bile were quantitated as described in the
Materials and methods. Each value represents the mean³SD from seven rats.
product, if formed in intact rat and excreted in bile, shows the same retention as
MO-GSH on hplc. Therefore, we analysed the reaction products obtained after
similar incubation conditions as reported by Correia et al. (1984a). This incubation
±
procedure produced two unknown metabolites with retention times of 7 5 and
±
8 0 min in addition to normorphine (retention time of 14 0 min), but a peak was not
±
±
observed at retention time of 12 0 min (chromatogram not shown). These two
unknown metabolites were not formed in the absence of glutathione in the
incubation mixture. These ®ndings indicate that two glutathione conjugated
metabolites, one of which probably is 10a-S-glutathionylmorphine, were formed
during the incubation but were separated from MO-GSH on hplc. Accordingly, we
conclude that the metabolite with retention time of 12 min in ®gure 3B is MO-GSH.
Figure 4 shows the cumulative biliary excretion of morphinone and MO-GSH
after s.c. injection of morphine (25 mg kg). The excretion of morphinone and MO-
}
GSH occurred very soon after morphine injection and was complete after 8 h. The
amounts of morphinone and MO-GSH excreted into the bile after 12 h were
±
±
±
±
0 8³0 3 and 8 2³4 3 % of the administered dose (n ¯ 7), respectively. Although the
amounts of both metabolites excreted in the bile varied between individual rats, the
amount of MO-GSH was 8±9 times that of morphinone in all rats.
In vitro formation of morphinone from morphine in rat, guinea pig, rabbit, mouse,
hamster and bovine liver
Figure 5 illustrates the morphine 6-dehydrogenase activity responsible for the
formation of morphinone from morphine in rat, guinea pig, rabbit, mouse, hamster
and bovine liver using the 9000 g supernatant as the enzyme source. Morphinone
formation was quantitated by hplc as the stable MO-ME derivative and the enzyme
activity was expressed as lmol morphinone formed per g liver per 30 min. All
species were capable of forming morphinone from morphine in the presence of
+
+
either NAD or NADP , although to diåering extents. Guinea pig showed the
+
highest activity with either NAD or NADP and the activities with either cofactor
+
+
were similar. In the rat, the activity with NAD was comparable with that in the
+
+
guinea pig but NADP supported about one-tenth of activity with NAD . Similarly,

650
S. Yamano et al.
Figure 5. Morphinone formation by guinea pig, rat, bovine, rabbit, mouse and hamster liver 9000g
supernatants. Incubation and quanti®cation of morphinone formed were carried out as described
in the Materials and methods. Data represent the mean of two-to-®ve animals except the mouse.
Values for the mouse are the mean of three diåerent preparations, each of 10 mice.
Figure 6. Subcellular distribution of morphine 6-dehydrogenase activity in guinea pig, rat, bovine,
rabbit, mouse and hamster liver. Incubations and quanti®cation of morphinone formed were
carried out as described in the Materials and methods. Data represent the mean of two-to-®ve
animals except mouse. Values for the mouse are the mean of three diåerent preparations, 10 mice
each.
+
the other four species exhibited a preference for NAD over NADP as cofactor.
+
+
Comparing the activity with NAD , rat and guinea pig gave three-to-four times
higher activity than bovine, rabbit, mouse and hamster.
The 9000 g supernatant was divided into the cytosolic and microsomal fractions
and subcellular distribution of the enzyme activity was examined (®gure 6). No
consistent subcellular distribution of enzyme activity was observed. In rat, enzyme
activity was almost exclusively distributed in the microsomal fraction, whereas the
enzyme activities in the guinea pig, bovine and hamster were localized mainly in the
cytosolic fraction. Rabbit and mouse exhibited higher activity in the cytosolic and
microsomal fractions respectively, but both fractions of these two species seemed to
contribute to the formation of morphinone from morphine in the liver.

Morphinone formation in the rat
651
Figure 7. Developmental changes of morphine 6-dehydrogenase activity in male and female rat liver
+
microsomes. Incubations using NAD as a cofactor and quanti®cation of morphinone formation
were carried out as described in the Materials and methods. Values represent the mean³SD of
!
three-to-seven male (E ) and female (D ) rats. * Signi®cantly diåerent from the female rat (p
±
0 05).
Figure 8. Eåects of pH on morphine 6-dehydrogenase activity in male (E ) and female (D ) rat liver
microsomes. Microsomes from either the adult male or female rat were incubated with morphine
+
(3 3 m ) and NAD (8 3 m ) in various buåers (pH 6 8±10 5) for 30 min at 37 C. Buåers used
°
were 100 m sodium phosphate buåer, Tris-HCl buåer or glycine±NaOH buåer. Each value
±
±
±
±
m
m
m
represent the average of duplicate determinations.
Developmental pattern of microsomal morphine 6-dehydrogenase activity in rat liver
+
Age-related changes of the enzyme activity with NAD in the microsomal
fractions of the male and female rat from 3 to 12 weeks of age are shown in ®gure 7.
In the male rat, the enzyme activity at 3 weeks, which was about 25 % that at 12
weeks, increased progressively up to 7 weeks of age and thereafter stayed at an
almost constant level. The female rat showed a similar developmental pattern to the
male until 5 weeks of age, but plateaued thereafter. Thus the enzyme activities in
±
immature rats of both sexes were similar, but at adulthood the male showed 2±2 5
times higher activity than the female. Similar developmental patterns of enzyme
+
activity with NADP were observed (data not shown).
Properties of microsomal morphine 6-dehydrogenase activity in the adult rat
Since a sex diåerence in morphine 6-dehydrogenase activity was observed in the
adult rat, we compared some properties between the adult male and female. Figure
8 shows the pH dependency of the enzyme activities in the adult male and female rat.

652
S. Yamano et al.
Figure 9. Lineweaver±Burk plots of morphine 6-dehydrogenase activity against morphine in male (E )
and female (D ) rat liver microsomes. Incubations were carried out as described in the Materials
±
and methods, except that various concentrations of morphine (1 3±6 7 m ) were used. Velocity, v,
±
m
l
is expressed as mol morphinone formed g liver 30 min.
}
}
Table 1. Inhibition of morphinone formation from morphine in adult male and female rat liver
microsomes.
Inhibition (%)
Concentration
m
Inhibitor
(m
)
Male Female
±
Pyrazole
Barbital
1 0
2
28
98
81
78
31
9
87
94
91
88
93
85
92
54
27
9
27
94
81
77
23
10
91
94
95
88
90
93
93
51
26
±
1 0
±
0 1
Quercetin
Naloxone
±
1 0
±
1 0
Indomethacin
Ketamine
Metyrapone
Testosterone
±
1 0
±
1 0
±
0 05
±
a
5 -Dihydrotestosterone
0 05
±
b
5 -Dihydrotestosterone
0 05
±
0 05
Androsterone
Epiandrosterone
Estradiol
Lithocholic acid
Chenodeoxycholic acid
Cholic acid
±
0 05
±
0 05
±
0 05
±
0 05
±
0 05
Incubations were carried out as described in the Materials and methods. Each value represents the
average of duplicate determinations. The amounts of morphinone formed with male and female rat liver
±
±
microsomes in the absence of inhibitor were 6 03 and 3 12 mol g liver 30 min respectively.
l
}
}
±
The maximum activity was obtained around pH 9 3 in both sexes, and the
activity±pH curves were quite similar. K for morphinone formation were estimated
m
by Lineweaver±Burk analysis and was determined to be 2 9 mm in the male adult
±
±
and 3 3 mm in the female adult rat. V
in the male was about twice that of the
max
female (®gure 9).
We ®nally compared the eåects of various compounds on the enzyme activities
in both sexes. As shown in table 1, all chemicals tested showed similar inhibitory
eåects on male and female enzyme activities. Androgens and estradiol inhibited
±
enzyme activity by about 90 % at 0 05 mm concentrations. Lithocholic acid, a
monohydroxyl bile acid, was also a potent inhibitor, but the inhibitory potency of
bile acid decreased as the number of hydroxyl groups in the molecule increased, at

Morphinone formation in the rat
653
least at the concentrations tested. Quercetin (inhibitor of aldo-keto reductase)
±
almost completely inhibited at 0 1 mm , but pyrazole (inhibitor of alcohol de-
hydrogenase) and barbital (inhibitor of aldose and alehyde reductase) had little or no
eåect on the enzyme activity. These eåects were similar to those previously reported
with guinea pig morphine 6-dehydrogenase puri®ed from the liver cytosol (Yamano
et al. 1985). Naloxone and indomethacin inhibited about 80 % at 1 mm.
Discussion
Metabolism of morphine has been extensively studied in various animals and
man, and consequently a number of metabolites have been identi®ed by in vivo and
in vitro experiments (Milne et al. 1996 and references therein). Although the pattern
of morphine metabolism is species-dependent, the main rout is commonly
glucuronidation to form M-3-G. In rat, the amount of M-3-G excreted in the 24-h
urine after morphine injection has been estimated to be 20±50 % of the dose (Klutch,
±
1974, Yeh et al. 1977, Kuo et al. 1991), and 2±6 and 1 5±4 % of dose have been also
recovered as normorphine including its 3-glucuronide and dihydromorphinone
respectively (Klutch 1974, Yeh et al. 1977). In addition to these latter metabolites,
M-6-G, a-dihydromorphine, and mono- and dihydroxymorphine have been iden-
ti®ed in the urine as minor metabolites (Yeh et al. 1979). In the present study, we
identi®ed both morphinone and MO-GSH in the bile of rat after s.c. administration
of morphine (25 mg kg) by hplc procedures using authentic standards for
}
±
±
comparison and also demonstrated that morphinone and MO-GSH (0 8³0 3 and
±
±
8 2³4 3 % of dose respectively) were excreted in the 12-h bile samples. However,
we could not detect neither morphinone nor MO-GSH in the urine (data not
shown). These results imply that rat produced morphinone in vivo accounting for
3±13 % of administered dose, and the majority (about 80±90 %) was conjugated with
glutathione and eliminated into the bile. This excretion is similar to that reported in
±
the guinea pig (Kumagai et al. 1990), in which 10 6 % of dose was excreted in the bile
as total morphinone, of which about 90 % was MO-GSH. The present study is the
®rst case in which in vivo formation of morphinone and MO-GSH in rat has been
demonstrated.
Nagamatsu et al. (1986) reported that isolated rat hepatocytes metabolized
morphine to morphinone and MO-GSH in addition to M-3-G and normorphine.
Although quantitative data were not presented, it is likely that M-3-G and MO-
GSH are major metabolites by judging from the magnitude of the chromatographic
peaks. On the other hand, Aasmundstad et al. (1993) reported that isolated rat
hepatocytes formed large amounts of M-3-G, normorphine and normorphine 3-
glucuronide (NM-3-G), but other metabolites (MO-GSH and morphinone) were
not detected in their study. Evans and Shanahan (1995) also reported that isolated
perfused rat livers metabolized morphine to M-3-G, normorphine and NM-3-G
accounting for 73, 11 and 10 % respectively of eliminated morphine, and the
remaining 6 % was due to unidenti®ed metabolites. These latter two studies suggest
that metabolism of morphine in rat liver is mainly proceeded by glucuronidation and
N-demethylation. However, the amount of total morphinone (sum of morphinone
and MO-GSH) excreted in the bile presented in this study was equal to or greater
than that of total normorphine (sum of normorphine and NM-3-G) recovered in the
urine (Klutch 1974, Yeh et al. 1977). This fact together with the result of Nagamatsu
et al. (1986) indicates that the metabolism of morphine to morphinone may be a

654
S. Yamano et al.
route of secondary importance to the glucuronide formation in morphine met-
abolism. In this connection, we found that the rate of formation of morphinone was
about four times that of normorphine in the same rat liver microsomes (data not
shown).
We have also demonstrated that the livers of all species examined contained
morphine 6-dehydrogenase activity (highest in guinea pig and lowest in hamster
based on wet tissue weight). The rate of morphinone formation in rat liver was
comparable with that in guinea pig liver, and three-to-four times that in mouse,
rabbit, hamster and bovine livers. Surprisingly, it was found that the enzyme
activity in rat liver was almost exclusively localized in the microsomal fraction,
which was quite diåerent from other species, and that rats exhibited a marked
+
preference for NAD over NADP compared with other species.
+
We are currently purifying rat liver microsomal enzyme after solubilization by
non-ionic detergent (octa-ethyleneglycol mono n-dodecyl ether). Although the rat
enzyme has not yet been obtained as a homogeneous protein, morphine 6-
dehydrogenase activity and 17b-hydroxysteroid dehydrogenase activity co-eluted
with each other at all stages in the puri®cation procedure, suggesting that the rat
liver enzyme may be identical to 17b-hydroxysteroid dehydrogenase and physio-
logically involved in androgen metabolism. In this connection, strong inhibition by
androgens in the present study is considered to be a result of their competition with
morphine at the substrate binding site of the enzyme.
Sex-diåerentiated hepatic metabolism in rat has been described for a variety of
drugs and steroids (Skett 1988). In the present study, it was found that morphine 6-
dehydrogenase activities in the immature rat of both sexes were similar but, at onset
of puberty to adulthood, male rat microsomes formed morphinone at about twice
the rate of female rat microsomes. This sex diåerence was statistically signi®cant but
not pronounced compared with that in N-demethylation to normorphine in which
the male rat showed about 15±20 times higher activity than the female (Blanck et al.
1990). In contrast, no sex diåerence in morphine 3-glucuronide formation has been
reported (Rush et al. 1983). V
for morphine 6-dehydrogenase activity of the male
max
rat was about twice that of the female but the K toward morphine, pH dependency
m
and inhibitor sensitivity were essentially the same in both sexes. These ®ndings
suggest that appearance of sex diåerence in morphine 6-dehydrogenase activity in
rat may be due to the quantitative change of the enzyme, which would be regulated
by endocrine but not due to the expression of sex-speci®c isozymes.
The administration of large doses of morphine to mouse or rat resulted in the
depletion of glutathione in the liver (James et al. 1982, Nagamatsu et al. 1982a,
Correia et al. 1984b) and potentiated the liver injury induced by acetaminophen and
cocaine (James et al. 1982, Skoulis et al. 1989a). This decrease in glutathione content
has been considered to be due to the action of morphine within the central nervous
system via opioid receptors (James et al. 1988, Skoulis et al. 1989b) and or due to the
}
hepatic metabolism of morphine to reactive metabolites which attack hepatocellular
glutathione and protein (Correia et al. 1984a, Nagamatsu et al. 1986, Eklow-
Lastbom et al. 1986). Nagamatsu et al. (1986, 1992) provided convincing evidence
for the involvement of hepatic metabolism of morphine to morphinone in morphine-
induced hepatotoxicity using isolated rat hepatocytes. The formation of MO-GSH
from intermediate metabolite, morphinone, occurred along with depleted cellular
concentration of glutathione and with decreased viability of the hepatocytes, and
morphinone±protein adducts were detected, particularly when the hepatocytes were

Morphinone formation in the rat
655
damaged. They also showed that the addition of naloxone decreased the formation
of MO-GSH and prevented the morphine-induced hepatocyte toxicity. These
eåects of naloxone have been assumed to be due to the inhibition of the formation
of morphinone. This assumption was evidenced by the ®nding that naloxone acted
as a powerful inhibitor of rat liver microsomal morphine 6-dehydrogenase in the
present study.
Other than morphinone, it has been postulated that the reactive intermediates
generated by cytochrome P450-catalysed oxidation deplete glutathione content in
the liver (Correia et al. 1984a, Eklow-Lastbom et al. 1986). Indeed, Correia et al.
(1984a) isolated a morphine±glutathione adduct following the incubation of
morphine with liver microsomes prepared from the phenobarbital-treated rat in the
presence of glutathione and NADPH which was identi®ed as 10a-S-glutathio-
nylmorphine. We also detected two glutathione-conjugated metabolites produced
by microsomes in the presence of NADPH and glutathione. However, these two
metabolites formed in vitro do not appear to be excreted in the bile, since the peaks
corresponding to these metabolites were not observed (®gure 3B). Therefore, it is
suggested that morphinone may be the main species responsible for the decrease in
liver glutathione content.
In conclusion, the metabolism of morphine to morphinone occurs in the intact
rat and contributes at least in part to the decrease of glutathione content in the liver
through subsequent formation of MO-GSH. In addition, the fact that the livers of
all species examined contained morphine 6-dehydrogenase activity suggests that
this interesting metabolic pathway is a common route in mammalian species. The
assessment of this metabolic pathway in man remains to be clari®ed, which is of
clinical signi®cance as morphine is often administered in large dose for analgesic
purpose and concurrently with other drugs which may depend on glutathione for
detoxi®cation.
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