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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1987
References
1. Samuelsson B: Leukotrienes: A new group of biologically active compounds including SRS-A. Trends Pharmacol Sci
9:227, 1980.
6.
7.
2. Kulkarni PS and Srinivasan BD: Synthesis of slow-reacting
substance-like activity in rabbit conjunctiva and anterior uvea.
Invest Ophthalmol Vis Sci 24:1079, 1983.
8.
3. Kulkarni PS, Rodriguez AV, and Srinivasan BD: Human anterior uvea synthesizes lipoxygenase products from arachidonic
acid. Invest Ophthalmol Vis Sci 25:221, 1984.
9.
4. Kulkarni PS and Srinivasan BD: Eicosapentaenoic acid metabolism in human and rabbit anterior uvea. Prostaglandins
31:1159. 1986.
10.
5. Kulkarni P: Metabolism of arachidonic acid and eicosapentaenoic acid in ocular tissues of monkey, human and rabbit. In
International Symposium on Prostaelandin and Related
Vol. 28
Compounds in Ophthalmology, Nakijima A and Bazan NG,
editors. Tokyo, Juntendo University, 1986, p. 10.
Borgeat P and Samuelsson B: Metabolism of arachidonic acid
in polymorphonuclear leukocytes: Effects of the ionophore
A23187. Proc Natl Acad Sci USA 76:2148, 1979.
Bisgaard H, Ford-Hutchinson AW, Charleson S, and Taudorf
E: Detection of leukotriene G»-like immunoreactivity in tear
fluid from subjects challenged with specific allergen. Prostaglandins 27:369, 1984.
Higgs GA, Flower RJ, and Vane JR: A new approach to antiinflammatory drugs. Biochem Pharmacol 28:1959, 1979.
Bhattacherjee P, Hammond B, Salmon JA, Stepney R, and
Eakins KE: Chemotactic response to some arachidonic acid
lipoxygenase products in the rabbit eye. Eur J Pharmacol
73:21, 1981.
Kulkarni PS, Ford-Hutchinson AW, and Srinivasan BD: Leukotriene B4 in rabbit paracentesis and endotoxin-uveitis
models. ARVO Abstracts. Invest Ophthalmol Vis Sci 27
7. 1986.
Nicotinamide N-Oxide Reductase Activity in Bovine and Rabbit Eyes
Shigeaki Shimada,* Hiromu Mishima,* Shigeyuki Kiramura,t and Kiyoshi Tarsumif
The nicotinamide N-oxide reductase activity of a variety of
ocular tissues was investigated. The 9,000g supernatant of
ciliary body, retinal pigment epithelium-choroid, iris, retina
and cornea, but not lens, exhibited reductase activity under
anaerobic conditions when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. Among
these tissues, the highest activity was observed with ciliary
body. When the 9,000g supernatant of ciliary body was
fractionated, the 2-hydroxypyrimidine-linked reductase activity was mainly associated with the cytosolic fraction and
was markedly inhibited by menadione, an inhibitor of aldehyde oxidase. Similarly, in the presence of 2-hydroxypyrimidine, the cytosolic fraction of rabbit ciliary body exhibited nicotinamide N-oxide reductase activity which was susceptible to inhibition by menadione. These facts strongly
suggest that aldehyde oxidase present in mammalian eyes is
involved in the reduction of nicotinamide N-oxide to nicotinamide. Invest Ophthalmol Vis Sci 28:1204-1206, 1987
Nicotinamide N-oxide has been recognized as an
in vivo metabolite of nicotinamide,1 which is a well
known precursor of nicotinamide-adenine dinucleotide (NAD+) in animals. On the other hand, the metabolic reduction of other N-oxide compounds has also
been reported. Chaykin and Bloch1 were the first to
demonstrate that nicotinamide N-oxide is reduced to
nicotinamide by hog liver homogenate. An enzyme
capable of catalyzing the N-oxide reduction was isolated from hog liver and identified as xanthine oxidase by Murray and Chaykin.2 Recently, Kitamura and Tatsumi3 found that aldehyde oxidase
(EC 1.2.3.1) rather than xanthine oxidase is the major
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enzyme responsible for the reduction of nicotinamide
N-oxide in mammalian livers. However, no reports
are available which describe the metabolic reduction
of nicotinamide N-oxide in extrahepatic tissues. The
aldehyde oxidase-dependent reaction is as follows: 2hydroxypyrimidine + nicotinamide N-oxide —• uracil + nicotinamide.
We report here the first description of tissue localization of nicotinamide N-oxide reductase activity in
the eye. We demonstrate the participation of aldehyde oxidase in the N-oxide reduction catalyzed by
ciliary body, retinal pigment epithelium-choroid, iris,
retina and cornea preparations.
Materials and Methods. Tissue preparation: Fresh
bovine eyes were obtained from a local slaughterhouse and brought to the laboratory on ice. Rabbit
eyes were removed from albino rabbits in the laboratory just prior to use. Cornea, lens, iris, ciliary body,
retina and retinal pigment epithelium-choroid were
dissected out separately. Subcellular fractionation of
ocular tissues was performed with a modification of
the method reported by Das and Shichi,4 as follows:
the tissue was homogenized with four volumes of
0.02 M Tris-HCl buffer (pH 7.4) containing 0.25 M
sucrose, first in a Polytron and then in a Potter-Elvehjem homogenizer. The homogenate was then
centrifuged for 20 min at 9,000g. In the case of ciliary
body, the 9,000g supernatant was further centrifuged
for 60 min at 105,000g to separate the cytosol from
microsomes. The microsomes were washed by resuspension in the Tris-HCl buffer and resedimentation
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Reports
No. 7
Table 1. Nicotinamide N-oxide reductase activity of 9,000g supernatants from a variety of bovine ocular tissues
Nicotinamide formed (pmol/min/mg
protein)
Addition
Lens
Cornea
Retina
RPE*-choroid
Iris
Ciliary body
None
2-hydroxypyrimidine
NADPH
NADH
Xanthine
0
0
50
0
0
0
110
0
170
0
0
290
80
30
0
0
4,450
0
70
0
0
3,610
0
0
0
0
11,710
0
0
40
Each value represents the mean of four experiments.
* Retinal pigment epithelium.
The incubation mixture consisted of 0.5 ^mol of nicotinamide N-oxide, 2
Mmol of an electron donor and the sample tissue preparation in a final volume of
1 ml of 0.1 M Tris-HCI buffer (pH 7.4). The mixture was incubated using a
Thunberg tube for 10-min at 37°C under an atmosphere of nitrogen; the reaction was stopped by adding 0.1 ml of 5N NaOH. A control tube lacking nicotinamide N-oxide was run with each reaction tube to correct for formation of
nicotinamide other than that from nicotinamide N-oxide. After adding 30 ^g of
benzamide as an internal standard and 0.1 g of NaCl, the mixture was extracted
twice with 5 ml each of ethyl acetate and the combined extract was evaporated to
dryness in vacuo. The residue was dissolved in 0.1 ml of methanol and then
subjected to high-pressure liquid chromatography (HPLC). HPLC was performed in a Toyo Soda HLC-803A chromatograph equipped with a UV-8 UV
absorption detector. The instrument was fitted with a 15-cm X 4.6-mm (I.D.) M
& S Pack Cl8 column. The mobile phase was methanol-water (15:85). The
chromatograph was operated at a flow rate of 0.80 ml/min at ambient temperature and at a wavelength of 254 nm. The nicotinamide (elution time 5.7 min)
formed was determined from its peak area.
for 60 min at 105,000g. All animals were treated in
accordance with ARVO Resolution on the Use of
Animals in Research.
Determination of protein: Protein content was determined by the method of Lowry et al5 with bovine
serum albumin as a standard.
Results. The ability to reduce nicotinamide Noxide to nicotinamide was examined with a varity of
bovine ocular tissues. As shown in Table 1, the
9,000g supernatant of ciliary body, retinal pigment
epithelium-choroid, iris, retina and cornea, but not
lens, exhibited nicotinamide N-oxide reductase activity under anaerobic conditions in varying degrees
when supplemented with 2-hydroxypyrimidine, an
electron donor of aldehyde oxidase. Among these tissues, the highest activity was observed with ciliary
body, followed by retinal pigment epithelium-choroid and iris. However, only slight or no reductase
activity was observed with NADPH, NADH or
xanthine, which is an electron donor for the cytochrome P-450 system, DT-diaphorase or xanthine
oxidase. On the other hand, the 9,000# sediment of
each tissue showed no ability to reduce nicotinamide
N-oxide even in the presence of the electron donors
described above.
When the 9,000g supernatant of ciliary body was
further fractionated by centrifugation, the 2-hydroxypyrimidine-linked reductase activity was
mainly associated with the cytosolic fraction and
markedly inhibited by menadione (1 X 10~4 M),
chlorpromazine (2 X 10"4 M) or amidol (2 X 10~4
M), which are inhibitors of aldehyde oxidase. The
cytosolic fraction also exhibited nicotinamide Noxide reductase activity, but to a lesser extent, when
supplemented with other electron donors of aldehyde
oxidase, such as N'-methylnicotinamide and benzaldehyde. However, little or no reductase activity was
observed with the microsomal fraction in the presence of NADPH or NADH.
The ability of rabbit ciliary body to reduce nicotinamide N-oxide was also examined in the same
manner. Its cytosol with 2-hydroxypyrimidine exhibited reductase activity comparable to that shown by
bovine ciliary body as described above. The activity
was again markedly inhibited by menadione (1
X 10"4 M).
These results strongly suggest that aldehyde oxidase present in mammalian eyes is involved in the
reduction of nicotinamide N-oxide.
Discussion. In the present study, no attempt was
made to purify aldehyde oxidase from ocular tissues
and directly examine its ability to reduce nicotinamide N-oxide. However, a recent study done by FCitamura and Tatsumi3 showed that rabbit liver aldehyde oxidase, in the presence of an electron donor
such as 2-hydroxypyrimidine and under anaerobic
conditions, exhibits a significant nicotinamide Noxide reductase activity which is markedly inhibited
by menadione. The enzyme is located in the liver
cytosolic fraction, which also exhibits N-oxide reductase activity when supplemented with an electron
donor specific for the enzyme. These facts support
the assumption that nicotinamide N-oxide reductase
activity detected in ocular tissues, especially in the
ciliary body, is mainly due to aldehyde oxidase.
Previously, Das and Shichi4 reported the highly
specific localization of some drug-metabolizing enzyme activities in bovine eyes: the highest activities of
aryl hydrocarbon hydroxylase and UDPglucuronosyltransferase were found in the ciliary body, followed by the retinal pigment epithelium-choroid and
iris. Mercapturic acid-synthesizing enzyme activities
were also associated with these tissues. The present
study, however, showed a similar localization of nic-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1987
otinamide N-oxide reductase activity in bovine eyes,
suggesting that the ocular tissues described above are
rich in enzyme activities responsible for not only drug
metabolism but also intermediary metabolism.
As regards aldehyde oxidase, only the liver enzyme
has been extensively characterized. Its molecular
weight has been estimated to be about 300,000. This
liver enzyme, which contains molybdenum, iron and
FAD in its molecule, can catalyze the oxidation of
both aldehydic and N-heterocyclic compounds, and
can utilize electron acceptors such as molecular oxygen and potassium ferricyanide. In the field of drug
metabolism, this liver enzyme has been recognized as
the major reductase responsible for the reduction of a
variety of xenobiotics, such as sulfoxides,6 nitrosoamines,7 azo dyes,8 N-oxides,3 aromatic nitro compounds,9 and hydroxamic acids.10
Key words: bovine, rabbit, eye, metabolic reduction, nicotinamide N-oxide, aldehyde oxidase
From the *Department of Ophthalmology and the "f Institute of
Pharmaceutical Sciences, Hiroshima University School of Medicine, Hiroshima, Japan. Submitted for publication: September 18,
1986. Reprint requests: Shigeaki Shimada, Department of Ophthalmology of Hiroshima University School of Medicine, 1-2-3,
Kasumi, Minami-ku, Hiroshima 734, Japan.
Vol. 28
References
1. Chaykin S and Bloch K: Metabolism of nicotinamide N-oxide.
Biochem Biophys Acta 31:213, 1959.
2. Murray KN and Chaykin S: The reduction of nicotinamide
N-oxide by xanthine oxidase. J Biol Chem 241:3468, 1966.
3. Kitamura S and Tatsumi K: Involvement of liver aldehyde
oxidase in the reduction of nicotinamide N-oxide. Biochem
Biophys Res Comm 120:602, 1984.
4. Das ND and Shichi H: Enzymes of Mercapturate synthesis and
other drug-metabolizing reactions: Specific location in the eye.
Exp Eye Res 33:525, 1981.
5. Lowry OH, Rosenbrough NJ, Fair AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem
193:265, 1951.
6. Yoshihara S and Tatsumi K: Guinea pig liver aldehyde oxidase
as a sulfoxide reductase: Its purification and characterization.
Arch Biochem Biophys 242:213, 1985.
7. Tatsumi K, Yamada H, and KJtamura S: Reductive metabolism of N-nitrosodiphenylamine to the corresponding hydrazine derivative. Arch Biochem Biophys 226:174, 1983.
8. Kitamura S and Tatsumi K: Azo reductase activity in liver
aldehyde oxidase. Chem Pharmacol Bull 31:3334, 1983.
9. Tatsumi K, Kitamura S, and Narai N: Reductive metabolism
of aromatic nitro compounds including carcinogens by rabbit
liver preparations. Cancer Res 46:1089, 1986.
10. Sugihara K and Tatsumi K: Participation of liver aldehyde
oxidase in reductive metabolism of hydroxamic acids to
amides. Arch Biochem Biophys 247:289, 1986.
Methotrexate-Anticollagen Conjugate Inhibits In Vitro Lens Cell Outgrowth
Thomsen J. Hansen,*t Roxane Tyndoll,-(- and David D. Soll^
After-cataract, or posterior lens capsule opacification, is an
undesirable but common sequela to extracapsular cataract
surgery. We are investigating biochemical means to prevent
after-cataract formation, which can be applied at the time of
the original surgery. Based on similar research efforts in
cancer chemotherapy, we have prepared a conjugate of the
antimetabolic agent methotrexate with an antibody specific
for basement membrane collagen, the major protein in the
lens capsule. The conjugate was evaluated using biochemical measurements, and retained both antimetabolic and antibody activities. When the conjugate was applied to bovine
posterior capsules in vitro, or in vivo in rabbits, it was an
effective inhibitor of lens epithelial cell outgrowth in cell
culture. Invest Ophthalmol Vis Sci 28:1206-1209, 1987
Extracapsular lens extraction has become the
method of choice for removing cataracts. The major
medical advantages of this technique over intracapsular extraction are lower incidences of both aphakic
cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber-type intraocular lenses,1
which are now considered to be the lenses of choice in
most cases. A disadvantage of extracapsular cataract
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extraction is the high incidence of posterior lens capsule opacification, often called after-cataract, which
can occur in up to 50% of cases within 3 yr after
surgery.2 After-cataract is caused by proliferation of
equatorial and anterior capsule lens epithelial cells
which remain after extracapsular lens extraction.3
This can be treated by additional surgery, but prevention of after-cataract would be preferable to treatment, and could be achieved by physically or chemically destroying the lens cells at the time of the original cataract extraction.
In animal experiments, cytotoxic drugs administered during surgery4 or intraperitoneally5 have been
investigated as a means of inhibiting lens epithelial
cells. Of these, the most successful has been methotrexate (MTX). MTX kills dividing cells preferentially, though not exclusively, and is used in cancer
chemotherapy.6 Because MTX is not specific as to
the type of cell that it kills, serious side effects can
occur. Targeting MTX to cancer cells has been attempted by covalently linking the drug to an antibody specific for that cell type.7 Targeting simultaneously allows a greater concentration of MTX in the