EPA-600/1-76-028
August 1976
Environmental Health Effects Research Series
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environ-
mental Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application
of environmental technology. Elimination of traditional grouping was con-
sciously planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS
RESEARCH series. This series describes projects and studies relating to the
tolerances of man for unhealthful substances or conditions. This work is gener-
ally assessed from a medical viewpoint, including physiological or psycho-
logical studies. In addition to toxicology and other medical specialities, study
areas include biomedical instrumentation and health research techniques uti-
lizing animalsbut always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-76-028
August 1976
THE PHARMACODYNAMICS OF CERTAIN ENDOGENOUS MAMMALIAN
ANTIOXIDANTS DURING N02 EXPOSURE
By
Kenneth D. Lunan and Alan E. Brandt
Environmental Biochemistry Laboratory
Stanford Research Institute
Menlo Park, California 94025
Contract No. 68-02-1713
Project Officer
George M. Goldstein
Clinical Studies Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
IT ":
l. '; -
* , ,,._MAL PROTECTION AStflft
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U. S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U. S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
11
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FOREWORD
The benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.
These studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants. The Laboratory develops and revises air
quality criteria documents on pollutants for which national ambient air
quality standards exist or are proposed, provides the data for registration
of new pesticides or proposed suspension of those already in use, conducts
research on hazardous and toxic materials, and is preparing the health
basis for non-ionizing radiation standards. Direct support to the
regulatory function of the Agency is provided in the form of expert
testimony and preparation of affidavits as well as expert advice to the
Administrator to assure the adequacy of health care and surveillance of
persons having suffered imminent and substantial endangerment of their
health.
The assessment of the relative risk of an environmental hazard
requires careful research and documentation of its adverse health related
effects. This report documents the antioxidant role of vitamin E in
preventing the peroxidation of polyunsaturated fatty acids by oxidizing
atmospheres such as nitrogen dioxide and ozone. These fatty acids are
constituents of cellular membranes, an alteration of which severely
affects biological processes.
Kneison, M.D.
Director,
Ileafth Effects Research Laboratory
ill
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Rats exposed to atmospheres containing nitrogen dioxide (N02)
in excess of 10 ppm showed a 50% increase in uptake of l4C-o/-
tocopherol by the lung when compared with control rats maintained
in ambient air. This increase was not observed in liver or
blood, the retention of l4C-a-tocopherol being the same in ex-
posed and control animals. NO- exposure did not affect the
half-life of l4C-a-tocopherol in lung, liver, or blood.
The liver of rats exposed to greater than 10 ppm N02 or to 1
ppm ozone showed a statistically significant (P < 0.05) increase
in the level of a-tocopherol oxidation products compared with
control rat liver, as judged by an increase in the ratio of
a-tocopherol quinone plus cv-tocopherol dimer to o'-tocopherol.
This increase was limited to the liver and was not observed in
either lung or blood. Liver, lung, and blood of vitamin E-
deficient rats exposed to 5 ppm N02 did not show any statistically
significant increase in O'-tocopherol oxidation products when
compared with control tissues. No effect of N03 on l4C-retinol
acetate metabolism was observed.
This research resulted in the first description of an enzyme
involved in a-tocopherol metabolism--namely, a UDP-glucuronic
acid:dihydro-a-tocopheronolactone glucuronosyl transferase, the
final enzyme in Q'-tocopherol metabolism before excretion. The
glucuronosyl transferase is a microsomal enzyme found predominantly
in the liver, and does not require a divalent cation for activity,
++ ++ ++
although it is stimulated by Sn , Ca , and Mg
This report was submitted in fulfillment of SRI Project LSU-3484,
Contract No. 68-02-1713, by Stanford Research Institute under
the sponsorship of the Environmental Protection Agency. Work was
completed as of October 21, 1975.
iv
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CONTENTS
Page
DISCLAIMER ii
FOREWORD iii
ABSTRACT iv
LIST OF FIGURES vi
LIST OF TABLES vii
LIST OF ABBREVIATIONS AND SYMBOLS viii
ACKNOWLEDGEMENTS xi
Sections
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV MATERIALS AND METHODS 9
V EXPERIMENTAL RESULTS 17
VI DISCUSSION 39
VII REFERENCES 51
TECHNICAL REPORT DATA 57
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FIGURES
No. Page
1 Disappearance of 1I+C-a-Tocopherol from Rat Lung ... 18
2 Disappearance of 1LfC-a-Tocopherol from Rat Liver . . 19
3 Disappearance of 1LfC-a-Tocopherol from Rat Blood . . 19
4 Total 14C-a-Tocopherol Content of Rat Lungs 21
5 Total 11+C-a-Tocopherol Content of Rat Livers .... 22
6 Thin-Layer Chromatography of lt+C-a-Tocopherol
and Metabolites from Lungs of NC^-Exposed
and Control Rats 24
7 Thin-Layer Chromatography of 11+C-a-Tocopherol
and Metabolites from Livers of N02~Exposed
and Control Rats 25
8 Ratio of a-Tocopherol Oxidation Procucts to
a-Tocopherol in Exposed and Control Rat Lungs .... 26
9 Ratio of a-Tocopherol Oxidation Products to
a-Tocopherol in Exposed and Control Rat Liver .... 26
10 Oxidant Gas Exposure Regimes 28
11 Thin-Layer Chromatography of -^C-Retinol Acetate
and Metabolites from Livers of N02~Exposed and
Control Rats 31
12 Formation of a-Tocopheronolactone Glucuronide
with Time 32
13 Formation of a-Tocopheronolactone GLucuronide
at Varying Protein Concentrations 33
14 Saturation of Glucuronosyl Transferase with
a-Tocopheronolactone 34
15 Saturation of Glucuronosyl Transferase with
UDP-GlcUA 34
16 Effect of N02 Exposure on Glucuronosyl Transferase
Activity in Monkey Liver 38
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TABLES
No. Page
1 Tissue Weight of NC»2-Exposed and Control Rats 20
2 Half-Life of lt+C-a-Tocopherol in Tissues of
N02-Exposed and Control Rats 23
3 Ratio of a-Tocopherol Oxidation Products to
a-Tocopherol in Tissues of Oxidant Gas-
Exposed and Control Rats 29
4 Effect of Divalent Cations on Glucuronosyl
Transferase Activity 36
5 Glucuronosyl Transferase Activity in Rat Tissues ... 37
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ABBREVIATIONS AND SYMBOLS
a
6
BHT
++
Ca
14C
C
cm
++
Co
-H-
Cu
cu ft
§
L
dpm
FADH2
v
GlcUA
>
hr
in
ip
kl
Alpha
Beta
Butylated hydroxytoluene
Calcium, divalent ion
Carbon 14
Celsuis
Centimeter
Cobalt, divalent ion
Copper, divalent ion
Cubic feet
Delta
Designates one of the two possible configurations
about,an asymmetric center
Disintegrations per minute
Flavin adenine dinucleotide, reduced form
Gamma
Gram
Gravity
Glucuronic acid
Greater than
Hour
Inch
Intraperitoneal
Kiloliter
Less than
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Mn Manganese, divalent ion
I j
Mg Magnesium, divalent ion
Km Michaelis constant
uCi Microcurie
l_i.g Microgram
til Microliter
umole Micromole
mg Milligram
ml Milliliter
mm Millimeter
mM Millimolar
min Minute
M Molar
nmole Nanomole
j i_
Ni Nickel, divalent ion
NADH Nicotinamide adenine dinucleotide, reduced form
NADPH Nicotinamide adenine dinucleotide phosphate, re-
duced form
MS Nitrogen
N0a Nitrogen dioxide
03 Ozone
ppm Part(s) per million
% Percent
P04 Phosphate, inorganic
p Probability
R Relative migration rate
sq. in. Square inch
[S] Substrate concentration
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TLC Thin-layer chromatography
_| |_
Sn Tin, divalent ion
UDP Uridine diphosphate
V Velocity, initial
v/v Volume per volume
-H-
Zn Zinc, divalent ion
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ACKNOWLEDGMENTS
The Environmental Biochemistry Laboratory of the Life Sciences
Division of Stanford Research Institute acknowledges the ex-
cellent technical assistance of Mrs. Jan Kilner in the per-
formance of all aspects of this work. We thank Dr. Gustave
Freeman, Director, Medical Sciences Department of the Life
Sciences Division, for helpful discussions and for making avail-
able the N02 and 03 exposure chambers. We also thank Dr. Free-
man for providing the samples of monkey liver.
We express our appreciation to Dr. William T. Colwell, Dr.
Joseph I. DeGraw, Mr. Vernon H. Brown, and Miss Caroline SooHoo
for the preparation of the l4C-a-tocopherol and -"-^C-retinol
acetate.
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SECTION I
CONCLUSIONS
The lung tissue of rats exposed to greater than 10 ppm N02
showed an approximately 50% increase in uptake of l4C-a-
tocopherol when compared with lung tissue of rats maintained
in ambient air. This increase was not observed for other
tissues, including liver and blood. N02 exposure did not
affect the half-life of l4C-Q'-tocopherol in lung, liver, or
blood.
The liver of rats exposed to 10 or more ppm N02 or to 1
ppm ozone showed a statistically significant (p < 0.05) increase
in the level of a-tocopherol oxidation products as compared
with control rat liver. This was determined by comparing the
ratio of a-tocopherol oxidation products (a-tocopherol quinone
plus Q'-tocopherol dimer) to a-tocopherol. Use of this technique
minimizes differences due to variable recoveries of o/-tocopherol.
The increase in the o'-tocopherol oxidation productsra-tocopherol
ratio for liver was not observed in either lung or blood at any
N02 concentration tested, nor was it observed in liver, lung,
or blood of vitamin E-deficient rats exposed to 5 ppm N02.
Apparently, N02 exposure (10 or more ppm) causes increased
oxidation of cv-tocopherol. The oxidation products are then
rapidly cleared from the tissue of origin and transported to
the liver for subsequent metabolism and excretion.
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No effect of N02 exposure on l4C-retinol acetate metabolism was
observed. However, because of the known lability of retinol
compounds to light and oxygen and the attendant problem of assay,
we cannot state with certainty that N02 exposure has any effect
on retinol acetate metabolism.
One of the enzymes involved in Q'-tocopherol metabolism--namely,
UDP-glucuronic acid:dihydro-a-tocopheronolactone glucuronosyl
transferase--was also investigated. This enzyme catalyzes the
transfer of glucuronic acid from UDP-glucuronic acid to reduced
a-tocopheronolactone, the final step in o'-tocopherol metabolism
before excretion. The enzyme is found predominantly in the liver,
with detectable levels also occurring in the kidney. Subcellular
distribution studies suggest it is found in the microsomes (100,000
X B pellet). The glucuronosyl transferase has a K of approximately
m
2.8 mM for o'-tocopheronolactone and 8 mM for UDP-glucuronic acid.
Because of the ease with which dihydro-a-tocopheronolactone is
oxidized, incubations were performed using a-tocopheronolactone
as substrate. We found that microsomes contain NADH:o(-
tocopheronolactone reductase, which produces dihydro-a-
tocopheronolactone. We also found that the liver of monkeys
undergoing long-term N02 exposure (9 years, 2 to 9 ppm N03)
shows a decreased level of glucuronosyl transferase activity.
This is contrary to what was expected: N02 should increase
a-tocopherol oxidation products, and we would expect to see in-
creased levels of enzymes involved in a-tocopherol metabolism.
Additional work is required to further substantiate this observa-
tion.
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SECTION II
RECOMMENDATIONS
1. Examine urine, bile, and faces for the presence of l4C-
labeled compounds derived from l4C-a-tocopherol.
2. Examine in more detail the kinetics of absorption, reten-
tion, and release of l4rC-o/-tocopherol in response to N02
exposures.
3. Investigate the nature and relative quantities of 1'C-o/-
tocopherol excretion products with particular emphasis
on dose-response relationship with N02 exposures.
4. Examine kidney and liver for enzymes capable of catalyzing
the formation of o'-tocopheronolactone glucuronide.
5. Further characterize the UDP-GlcUA:dihydro-a-tocopheronolactone
glucuronosyl transferase, particularly for substrate
specificity.
6. Characterize the enzymatic steps involved in the metabolism
of a-tocopherolquinone to a-tocopheronolactone.
7. Carry out further studies on l4C-retinol metabolism under
more stringent conditions of atmospheric control.
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SECTION III
INTRODUCTION
Vitamin E, a fat-soluble, antisterility factor, has been the sub-
ject of increasing research since its discovery by Evans and
Bishop1'2 50 years ago. Although the basis for its action as an
antisterility factor remains unexplained, numerous additional
biological functions have been ascribed to Vitamin E.
The predominant theory for vitamin E action is based on the
antioxidant properties of the vitamin. Vitamin E prevents the
peroxidation of polyunsaturated fatty acids found in lipids of
cellular membranes, thereby stabilizing membrane structure. This
action is demonstrated by the marked fragility of red blood cells
of animals deficient in vitamin E.3 Lipid peroxidation is initi-
ated by exposure to hyperbaric oxygen and other oxidizing atmo-
spheres such as nitrogen dioxide (NOg) and ozone (0-. ). Vitamin
E is believed to quench highly toxic free radicals generated
during peroxidation, thus terminating the free radical chain
reaction.4
Several investigators have seriously questioned this role of vita-
min E, principally regarding the quantitative relationships among
a-tocopherols, the degree of peroxidation, and the appearance
of lipid peroxides.5 Recent research demonstrating the metabolism
of lipid peroxides by glutathione peroxidase at the expense of
NADPH counters the last objection.6
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Other activities of vitamin E have been discovered. The vitamin
has been demonstrated to affect the biosynthesLs of heme by
activating the initial controlling enzyme, 6-aininolevulinic
acid synthetase.7 In this role, vitamin E is involved in deter-
mining levels of hemoglobin and, hence, the oxygen-carrying
capacity of red blood cells and levels of heme-containing
cytochrome, thereby affecting mitochondrLai electron transport
r*
and oxidative phosphorylation. In addition, vitamin E has been
reported to directly affect mitochondriaL oxygen consumption
and to be involved in microsomal drug metabolism9 and selenium
metabolism.5
Very little is known about the enzymatic process governing
O'-tocopherol metabolism. One postulate is that, once G^tocopherol
has been oxidized to Q'-tocopherolquinone, a sequence of enzymatic
transformations occurs that results in the degradation of the
isoprenyl side chain of Q'-tocopherol, yielding Q'-tocopheronolactone.
Stumpf1 has reviewed several postulated mechanisms, but enzymes
capable of carrying out the required reactions have not been
reported.
In 1912, Hopkins1 identified vitamin A as a necessary nutrient
required for normal growth. Subsequently, the olant pigment
carotene was found to be an effective precursor to vitamin A
that could be used as a dietary supplement in place of vitamin A.ls
Vitamin A is an extremely labile compound readily isomerized by
light and oxidized by oxygen. This sensitivity to light is the
basis for the biological role of vitamin A as a visual pigment
o
in association with rhodopsin. Rosso et al. demonstrated that
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vitamin A also serves as a lipid-phosphate intermediate in
certain glycosyl-transferase reactions.
Edwin et al.14 demonstrated that vitamin E has a protective
effect on vitamin A in* biological tissues by preventing its
oxidation. Therefore, because animals exposed to an oxidizing
gas such as N02 exhibit decreased levels of vitamin E, a reason-
able expectation is that secondary effects on vitamin A may
also be observed.
This report describes studies on the effect of N02 on the reten-
tion of l4C-o?-tocopherol in lungs, liver, and blood and on the
formation and disposition of o?-tocopherol and retinol oxidation
products. Also presented are initial results on the character-
ization of one of the enzymes involved in a-tocopherol metabolism,
UDP-glucuronic acid:a-tocopheronolactone glucuronosyl transferase.
In these studies, rats were continuously exposed to subacute
1 B 21
levels of N03 or 03 . Earlier studies ~ provided us knowledge
of the well-defined sequence of morphological events arising
from such exposure. During the first 24 hours, injury to and
loss of both ciliated cells from the bronchiolar epithelium
and Type I cells of the alveoli occur. Replacement of these
cells by division of nonciliated and Type II cells begins and
reaches a peak at the end of the second day. By the third day,
the accumulation of cellular debris, fibrinous exudate, and
macrophages and the hypertrophy of nonciliated cells cause
obstruction of the small airways. After about 7 days of
£
7
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continuous exposure, considerable repair has occurred, and the
lung assumes a more normal appearance. However_, further exposure
causes additional cellular changes and invariably leads to the
development of a disease resembling emphysema.
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SECTION IV
MATERIALS AND METHODS
PREPARATION OF l4C-a-TOCOPHEROL
To prepare a-tocopherol labeled with 1 C in the 5-methyl position,
3 2
we used the method of Nakamura and Kijima. Approximately
10 (aCi (specific activity, 10 (j.Ci/(jmole) of l4C-a-paraformaldehyde
was reacted with 150 mg of y-tocopherol. l4C-o/-tocopherol was
isolated from the reaction mixture by thin-layer chromatography
(TLC) on silica gel G plates in cyclohexane:ether (80:20, v/v).
The radioactive band corresponding to a-tocopherol was eluted
with ether, concentrated under N2, and stored in redistilled
benzene:ethanol (9:1, v/v) at -20°C until use. Before administra-
tion to rats, the l C-a-tocopherol was repurified by TLC as de-
scribed above. This procedure resulted in l4C-a-tocopherol
preparations of at least 98% radiopurity.
PREPARATION OF 14C-RETINOL ACETATE
To prepare 1~C-retinol acetate labeled in the 6-methyl group
of the (3-ionene ring, we used a combination of published
procedures.23"26 l4C-Methyl iodide was reacted with 2,6-dimethyl
cyclohexanone to yield 2,2,6-trimethyl cyclohexanone (2-methyl-
14C).2S 2,2,6-Trimethyl cyclohexanone was then condensed with
js-C
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? "-\ o e
3,7-dimethyl-4,6,8-nonatrien-l-yne-3-ol " " to yield 3,7-dimethyl-
l-(l-hydroxy-2,6,6-trimethyl-l-cyclohexyl)-3,5,7-nonatrien-l-yne-9-ol.
Subsequent reduction, acetylation, and dehydration yielded vitamin A
acetate.
RATS
We obtained male Sprague-Dawley rats, age 30 days, from Hilltop
Animal Farm, Scottdale, Pennsylvania. We chose this supplier
because its rats were free of lung mycoplasma and other respira-
tory diseases. The rats were housed either in a control room,
where they were exposed to ambient air, or in airflow chambers
having an internal volume of 2.34 kl (82.5 cu ft). The rats in
the airflow chambers were exposed to 5, 10, 14, or 20+1 ppm
N02 or to 1 ± 0.5 ppm 03. The rate of airflow through the
chambers was 1.13 kl/min (40 cu ft/min) at a slightly negative
pressure of 0.19 to 0.36 mm of mercury (0.1 to 0.2 in. of water)
below atmospheric pressure. Gas exposure chambers and generators
have been described previously.27->28 Rats were routinely fasted
for 12 hours before being administered l4C-cy-tocopherol or
T A
C-retinol acetate. Food was restored 4 hours postinjection.
ADMINISTRATION OF RADIOLABELED MATERIALS
l4C-a-Tocopherol
After exposure to N02 or 03, groups of five rats each and five
control rats were injected intraperitonea lly (ip) with 1. to
3 ,uCi of l4C-o'-tocopherol in 0.5 ml of a 5% Tween 80 solution
prepared under nitrogen with degassed water. In initial experi-
ments^ we injected 1 (J,Ci of 3H-L-Leucine with the -1 "C-cy-tocopherol
10
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to follow the rate of protein synthesis. The exposed rats were
returned to the NO^ or 03 chambers until sacrifice; control rats
were maintained in ambient air.
l4C-Retinol Acetate
We used a procedure similar to that for administration of 1~C-O'-
tocopherol to give rats ip injections of l4C-retinol acetate.
The only difference was that the procedure was carried out in a
photographic darkroom under red light. All solvents were deaerated
before use. Storage and chromatography of retinol acetate were
done under an argon atmosphere.
ASSAY FOR l4C-a-TOCOPHEROL UPTAKE AND LEVEL
OF OXIDATION PRODUCTS
All procedures were conducted in diminished light. Two methods
were used to kill rats. The first was by ip injection of approxi-
mately 0.1 ml/100 g of body weight of a sodium pentobarbital
solution (1 g/ml). This permitted the surgical exposure of the
abdominal aorta and subsequent removal of a 3-ml blood sample
with a heparinized syringe before organ removal. The second
method was by decapitation and draining of blood into a heparinized
centrifuge tube. The blood samples thus obtained were centrifuged
for 10 minutes in a clnii'.-il centrifuge, and the plasma was de-
canted. Plasma obtained in this manner was used for subsequent
analysis for l4C-a-tocopherol and its oxidation products.
After blood collection, the liver and lungs were removed and
immediately placed on ice. The assay method used depended on
11
js-C
-------
whether we were investigating the total uptake and loss of
a-tocopherol and metabolite or whether we were separating and
quantitating a-tocopherol, oxidation products, and metabolites.
Total a-tocopherol and metabolites were estimated in the follow-
ing manner: Approximately 3 g of tissue was homogenized in three
volumes of normal saline (blood was diluted 1:4 with saline), and
aliquots were assayed for protein. The aliquots were absorbed
on a 1 sq. in. piece of filter paper, dried, and extracted with
chloroformtmethanol (2:1, v/v). The lipLd extract was dried
under Ns, and the l4C-labeled content was quantitated by liquid
scintillation spectrometry. The tissue content of a-tocopherol
and a-tocopherol metabolites (14C-labeled material) is expressed
as disintegrations per minute (dpm) per milligram of protein
or as total dpm per organ.
To determine the level of oxidation products of a-tocopherol
(i-tocopherolquinone, a-tocopherol dimer and trimer), we used
the following procedure. Approximately 1 g of liver or lung
was finely minced and placed in the bottom of an 18 x 1-50 mm
test tube; 2 ml of a 2% pyrogallol solution in 95% ethanol and
2 ml of saturated potassium hydroxide were added. Samples were
hydrolyzed at 70°C for 30 minutes and cooled, 4 ml of water was
added, and samples were extracted twice with 4 ml of 1.25 mM
BHT in hexane. The combined hexane extracts were evaporated
to dryness under a stream of nitrogen, dissolved in 0.2 ml of
hexane, and subjected to TLC on silica gel G plates in cyclohexane:
ether (80:20, v/v). The plates were allowed to develop to
approximately 14 cm (in the dark). After evaporation of the
12
is-C
-------
solvent, the plates were scraped in 1-cm bands and placed in
scintillation vials; 5 ml of scintillation fluid was added, and
radioactivity was determined in a liquid scintillation counter.
After subtracting background, the total dpm corresponding to
a-tocopherol, a?-tocopherolquinone, a-tocopherol dimer, and
a-tocopherol trimer was determined, and the ratio of a-tocopherol
(dpm) to a-tocopherol oxidation products (dpm) was calculated.
ASSAY FOR 14C-RETINOL ACETATE UPTAKE AND DISPOSITION
IN TISSUES
Ten rats were exposed to 10 ppm of N02 for 3 days, injected
under red light with 1.25 |J,Ci of l4C-retinol acetate in deaerated
5% Tween 80, and returned to chambers containing 10 ppm N02.
Ten control rats were injected in the same manner and maintained
in ambient air. Groups of five exposed and five control rats
were killed at 24 and 72 hours postinjection. The liver, lungs,
and blood were then removed in a darkened room. Approximately 1 g
of each tissue was homogenized in one volume of distilled water,
followed by homogenization with 5 ml of ethanol. Vitamin A
was extracted from each tissue homogenate with cyclohexane
containing 1.25 mM BHT and centrifuged to separate phases. The
upper phase (cyclohexane) was removed and dried in vacuo in
total darkness. The concentrated organic extract was rapidly
spotted on silica gel G plates under red light in a darkroom
and subjected to TLC in an argon-filled tank in cyclohexane:
ether (80:20, v/v). After development, the thin-layer plates
were dried and divided into 1-cm sections, each of which was
13
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scraped into scintillation vials and counted for radioactivity
by liquid scintillation spectrometry.
ASSAY OF UDP-GLUCURONIC ACID:DIHYDRO-a-TOCOPHERONOLACTONE
GLUCURONOSYL TRANSFERASE
O g
We used the method of Graham and Wood to prepare rat liver
microsomes. Freshly excised rat liver was homogenized in 10
volumes of cold 0.25 M sucrose far 40 seconds in a Waring
blender. After centrifugation at 10,000 x .£_ f°r 20 minutes,
the supernatant was centrifuged at 80,000 x £ £°r 90 minutes.
The pellet was homogenized in 2 ml of 0.25 M sucrose per gram
of fresh liver and centrifuged at 80,000 x .£. as above. The
pellets were homogenized in 0.5 ml of- 0.154 M of potassium
chloride per gram of fresh liver and stored frozen in small
aliquots. For the tissue distribution study, we obtained a
crude enzyme preparation by homogenizing tissue in a Potter-
Elvehjem apparatus with two volumes of 0.25 M sucrose.
Typical incubations contained the following materials in a total
volume of 50 u,l: a-tocopheronolactone (2.5 jjmoles), NADH (5
M,moles), imidazole, pH 7.0 (5 p.moles), UDP-l4:C-GlcUA (1 pmole),
and microsomal enzyme protein (400 p,g) or crude tissue homogenate
(25 |J.l). After incubation at 37°C for 1 hour, 40 ul of the
reaction mixture was spotted on Whatman 3-rrtm paper and chro-
matographed in ethanol:! M ammonium acetate, pH 7.5 (7:3 v/v).
cy-Tocopheronolactone glucuronide migrated with an R of approxi-
mately 0.79 and was distinctly separatee from UDP-GlcUA, GlcUA,
*
and GlcUA-l-P04. The region containing dihydro-a-tocopheronolactone
14
js-C
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glucuronide was cut out, and radioactivity was quantitated by
liquid scintillation spectrometry.
SCINTILLATION FLUID
»
We prepared scintillation fluid by dissolving 22.74 g of PPO
and 274 mg of POPOP per gallon of reagent-grade toluene.
PROTEIN
We used the method of Lowry et al. to determine protein.
15
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SECTION V
EXPERIMENTAL RESULTS
UPTAKE AND LOSS OF a-TOCOPHEROL '
Figures 1, 2, and 3 show the retention of l4C-Q'-tocopheroL by
the lungs, liver, and blood, respectively, of rats exposed to
14 ppm N02 for 3 days and of control rats exposed to ambient
air. Both lungs and livers of N02-exposed animals retained
more l4C-cy-tocopherol per gram of tissue protein than did those
of controls. However, as indicated in Table 1, the lungs of
N02-exposed animals were larger than those of control rats be-
cause of the incipient development of a disease resembling
emphysema. Figure 4 presents the total l4C-o?-tocopherol content
of the rat lungs. This determination shows that the lungs of
N02-exposed rats, compared with those of control rats, retained
a significantly greater amount of l4C-a-tocopherol. In contrast,
the livers of N02-exposed animals were smaller than those of
controls (Table 1) so that, when the total -"C-a-tocopherol
content of liver was calculated, no difference between N02-
exposed and control animals was observed as shown in Figure 5.
The content of -1 C-cv-tocopherol in the blood of exposed and
control animals was the same at all times.
The rates of disappearance of l4C-Q'-tocopherol from lungs,
liver, and blood did not differ significantly between control
and exposed rats (Figures 1, 2, and 3).
17
js-C
-------
1000
800
600
400
z
LU
200
I 100
T>
80
60
40
30
NOTE: Brackets represent ±1_
standard deviation.
T1/2 Control 75.5 hrs
O Exposed to 14 ppm N02 for 3 Days
D Control
_J I I I
24 48 72 96 120
TIME AFTER ADMINISTRATION OF
14C-o-TOCOPHEROL hours
FIGURE 1 DISAPPEARANCE OF 14C-a-TOCOPHEROL
FROM RAT LUNG
Twenty rats were exposed to 14 ppm of N02
for 3 days and twenty rats were maintained in
ambient air as controls as described in Materials
and Methods. After 3 days, each rat received an
ip injection of 3 pCi of 14C-a-tocopherol
(specificity, 10 /jCi//umole) suspended in aqueous
5% Tween 80. Exposed rats were returned to
the chambers, and NO2 exposure was continued.
At the indicated time, five rats each from ex-
posed and control groups were killed, and the
total content of a-tocopherol and metabolites
was determined.
As indicated in Table 2, comparison of the half-life of l4C-a-
tocopherol in N03-exposed and control lung, liver, and blood
shows that the a-tocopherol is removed from the blood approxi-
mately twice as fast as it is from either the lungs or the
liver. No significance is attached to the small difference
observed in the half-life of x~C-o>tocopherol in NO--exposed
18
-------
1000
800
600
rfi 400
o
cr
Q.
200
100
80
60
NOTE- Brackets represent ±1
standard deviation.
T1/2 Exposed 79 hrs
T1/2 Control 78.5 hrs
O Exposed to 14 ppm NO2 for 3 Days
D Control
24 48 72 96
TIME AFTER ADMINISTRATION OF
14C-a-TOCOPHEROL hours
120
FIGURE 2 DISAPPEARANCE OF 14C-a-TOCOPHEROL
FROM RAT LIVER
Conditions were the same as those described
in Figure 1.
200
100
80
£ 60
LU
g 40
Q.
I 20
10
8
T
T1/2 Exposed 40 hrs
T.|/2 Control 37 hrs
O Exposed to 14 ppm N02 for 3 Days
~ O Control
I I I
24 48 72 96
TIME AFTER ADMINISTRATION OF
14C-a-TOCOPHEROL hours
120
FIGURE 3 DISAPPEARANCE OF 14C-a-TOCOPHEROL
FROM RAT BLOOD
Conditions were the same as those described
in Figure 1.
19
-------
Table 1. TISSUE WEIGHT OF N02-EXPOSED
AND CONTROL RATSa
(grams)
Tissue
Lung
N02 exposed
Control
Liver
N02 exposed
Control
24 hr
1.33 ± 0.19b
0.92 ± 0.08
p < 0.005°
6.12 - 0.90
8.57 ± 0.93
p < 0.005
48 hr
1.33 ± 0.15
1.06 ± 0.05
p < 0.01
5.51 ± 0.56
7.49 ± 0.50
p < 0.001
r 72 hr
1.48 ± 0.14
0.97 ± 0.05
p < 0.001
5.52 ± 0.47
7.35 ± 0.90
p < 0.005
96 hr
1.44 - 0.04
1.11 r 0.06
p < 0.001
5.47 r 0.71
7.96 = 0.27
p < 0.005
Rats were exposed to 14 ppm of N02 for 3 days before administration
of 3 (j.Ci of l4C-a-tocopherol in 5°L Tween 80. Exposure was con-
tinued until sacrifice at the indicated times following -~C-z<-
tocopherol administration.
Tissue weight r standard deviation.
'Standard Student's t-test.
and control lung tissue. Exposure to lower levels of NOS produced
even smaller differences between exposed and control groups.
EFFECT OF N02 EXPOSURE ON TISSUE LEVELS OF CV-TOCOPHEROL
OXIDATION PRODUCTS
To determine whether N02 exposure affected tissue levels of
a-tocopherol oxidation products^ we injected rats previously
20
-------
15
v°
1 5
a
O
D Control
I
"S,
Exposed to 14 ppm NO for 3 Days
24
48
72
96
TIME AFTER ADMINISTRATION OF
14
120
C-a-TOCOPHEROL hours
FIGURE 4 TOTAL 14C-a-TOCOPHEROL CONTENT OF RAT
LUNGS
Conditions were the same as those described in
Figure 1.
exposed to 14 ppm N0£ ip with 3 pCi of l4C-a-tocopherol and con-
tinued the N02 exposure. At 2k, 48, 12, and 96 hours after
1 "C-c^-tocopherol administration, we killed groups of five rats
and removed lungs; liver, and blood as described in Materials
and Methods. After TLC of the nonsaponifiable fraction, we
determined the relative amounts of radioactivity corresponding
to c^-tocopherol, o'-tocopherolquinone,, and Q'-tocopherol dimer.
Because cv-tocopherol dimer and a-tocopherol trimer have similar
R values in the solvent system used, and since little trimer
was found as determined by mass spectrometry, only a-tocopherol
dimer is reported.
21
is-C
-------
100
80
o 60
en
LU
40
20
O Exposed to 14 ppm NO for 3 Days
3 Control
24
48
72
120
TIME AFTER ADMINISTRATION OF
14C-a-TOCOPHEROL hours
FIGURE 5 TOTAL 14C-o:-TOCOPHEROL CONTENT OF RAT
LIVERS
Conditions were the same as those described in
Figure 1.
Figures 6 and 7 present typical results from lung and liver -;y-
rracts from NOS -exposed and control rats. o--Tocopherol;
:;-tocopherolquinone, and the dimer were readily separated fro--
each other. A variable peak of radioactivity (up to 107, of
the total) remained at the origin. This material is probabi,
_:.r. oxidation product of a-tocopherol formed nonenzymatica] !y
during saponif ication; it was also formed when pure l
wjis subjected to base hydrolysis under the conditions
C-v-to'-_ -, ;
22
-------
Table 2. HALF-LIFE OF l4C-o>TOCOPHEROL
IN TISSUES OF N02-EXPOSED AND CONTROL RATSa
(hours)
Tissue
Lung
Liver
Blood
N02 exposed
84
79
40
Control
76
78
37
Half-lives for lung, liver, and blood were
determined from data in Figures I, 2, and 3.
The length of time required for the level of
1 "C-cv-tocopherol to decline by 50% was deter-
mined from those data.
Its formation was minimized by including pyrogallol in the
hydrolysis medium as an antioxidant. For these reasons, we
included the radioactivity in the origin material with that of
the o'-tocopherol when we calculated the relative levels of
tissue cv-tocopherol. To circumvent the potential low recoveries
in a-tocopherol assays, we determined the ratio of c^-tocopherol
oxidation products to a-tocopherol as a function of NOg exposure.
When the data were expressed as the ratio of l4C-a-tocopherol
oxidation products (o'-tocopherolquinone plus a-tocopherol dimer)
to total l4C-a-tocopherol (origin material plus &-tocopherol),
no statistically significant difference in the ratio of cv-tocopherol
oxidation products to a-tocopherol was found in lung tissue.
23
js-C
-------
4000
o. 2000
.' ' : .
'.'.'.' ".'
;; "r.\.
FI^
U. ........ .r^r^| t
ORIGIN
a-TOCOPHEROL
a-TOCOPHEROL-
QUINONE
Q-TOCOPHEROL
DIMER
SOLVENT
FRONT
(a) LUNG EXPOSED TO 14 ppm NO,2 FOR 3 WEEKS
Z3UU
1500
a
T3
1000
500
:^-l:-
v;. -^y.
Q:
^m
'.-. ''<"'.'
W^U^^V-^T^
ORIGIN a-TOCOPHEROL
a-TOCOPHEROL- SOLVENT
QUINONE FRONT
(b) LUNG CONTROL
FIGURE 6 THIN-LAYER CHROMATOGRAPHY OF 14C-a-TOCOPHEROL AND
METABOLITES FROM LUNGS OF N02-EXPOSED AND CONTROL RATS
Figure 8 shows these data. In contrast,, a significantly higher
ratio was observed in livers of rats exposed to N02 as compared
with control rats, as shown in Figure 9. The proportion of
oxidation products in the liver increased with time in both
24
-------
8000
F
Q.
6000
4000
2000
''"''4. [., , .
: '.'''':;
['" 1
ORIGIN
a-TOCOPHEROL
a-TOCOPHEROL-
QUINONE
SOLVENT
FRONT
a-TOCOPHEROL
DIMER
(a) LIVER EXPOSED TO 14 ppm NO2 FOR 3 WEEKS
''£&
'.'.',:.-.-
I!
^iiLrMzM^
ORIGIN a-TOCOPHEROL SOLVENT
FRONT
a-TOCOPHEROL- a-TOCOPHEROL
QUINONE DIMER
(b) LIVER CONTROL
FIGURE 7 THIN-LAYER CHROMATOGRAPHY OF 14C-a-TOCOPHEROL AND
METABOLITES FROM LIVERS OF N02-EXPOSED AND CONTROL RATS
25
-------
1.6
1.4
1.2
_i
§ 1.0
LU
X
8 °-8
o
i 0.6
u
0.4
0.2
0
O Exposed to 14 ppm NCL for 3 Weeks
D Control
NOTE: Brackets, represent ±1
standarc deviation.
24 48 72 96
TIME AFTER ADMINISTRATION OF
14C-a-TOCOPHEROL hours
120
FIGURE 8 RATIO OF a-TOCOPHEROL IN EXPOSED AND CONTROL
RAT LUNGS
UJ
I
Q_
O
O
o
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
O Exposed to 14 ppm N02 for 3 Weeks
D Control
NOTE:
Brackets represent ±1
standard deviation.
24
48
72
96
120
TIME AFTER ADMINISTRATION OF
14C-a-TOCOPHEROL hours
FIGURE 9 RATIO OF a-TOCOPHEROL OXIDATION PRODUCTS IN
EXPOSED AND CONTROL RAT LIVER
26
-------
m
exposed and control rats, although the rate of accumulation was
somewhat greater in livers from N02-exposed rats.
As shown in Figure 10, we investigated the effects of different
concentrations of N02 and of different exposure regimes on the
ratio of a-tocopherol oxidation products to cv-tocopherol in
lungs, liver, and blood of the rat. Table 3 presents these
data. One exposure experiment with 03 was also performed. In
all these exposure experiments, N02 and 03 had no significant
effect on the proportion of Q'-tocopherol oxidation products in
the lungs. In blood, only the highest level of N02 used, 20 ppm,
significantly increased the oxidation products.
In contrast, the livers had significantly elevated levels of
oxidation products in all experiments except those in which the
lowest level of N02, 5 ppm, was used. The proportion of oxida-
tion products in livers from rats in the 10-day to 3.5-week
exposure experiments was much higher than that in livers from
animals exposed for 3 days. However, the levels of oxidation
products in livers from animals exposed to 10 and 14 ppm N02
for about 3 weeks were higher than those in the corresponding
controls. Only tissue from animals exposed to 5 ppm N02 had
levels of oxidation products that did not differ significantly
from the controls, even though these animals were deficient in
vitamin E.
We conducted one experiment with exposure to 1 ppm 03 for 3 days
before l C-a-tocopherol administration. Table 3 shows that
oxidation products were significantly high in the liver but not
27
js-C
-------
4. 14 ppm NO2
5. 10 ppm N02
A Time of 14C-a-Tocopherol Injection
A Time of Sacrifice
Period of Exposure to Oxidant Gas
Period of Administration of
Vitamin E-Deficient Diet
FIGURE 10 OXIDANT GAS EXPOSURE REGIMES
Experiments are identified and described in detail in the lootnote to Table 3 (p. 27).
28
-------
Table 3
RATIO OF a-TOCOPHEROL OXIDATION PRODUCTS TO a-TOCOPHEROL
IN TISSUES OF OXIDANT GAS-EXPOSED AND CONTROL RATSa
Experiment
No.
1
2
3
4
5
6
Initial
Exposure
1 ppm 03
3 days
20 ppm N02
3 days
14 ppm N02
3 days
14 ppm N02
3 weeks
10 ppm N02b
3-1/2 weeks
5 ppm N02
10 days
Animal
Groups
Exposed
Control
Exposed
Control
Exposed
Control
Exposed
Control
Exposed
Control
Exposed
Control
Lung
0.40 ± 0.20
0.24 ± 0.12
p > 0.2
0.81 ± 0.32
0.48 ± 0.28
p > 0.05
0.53 ± 0.18
0.18 ± 0.07
p < 0.025
0.66 ± 0.08
0.54 ± 0.10
p > 0.1
0.63 ± 0.13
0.65 ± 0.20
P > 0.5
0.63 + 0.10
0.59 ± 0.12
p > 0.5
Liver
0.50 ± 0.15
0.26 ± 0.05
p < 0.05d
0.34 ± 0.15
0.15 ± 0.06
p < 0.05
0.31 ± 0.13
0.11 ± 0.03
p < 0.01
1.29 ± 0.12
0.74 ± 0.22
p < 0.005
1.03 ± 0.17
0.69 ± 0.15
p < 0.05
0.91 ± 0.39
1.24 ± 0.40
p > 0.4
Blood
0.39 ± 0.18
0.44 ± 0.23
p > 0.5
0.34 ± 0.19
0.14 ± 0.04
p < 0.05
0.47 ± 0.22
0.34 ± 0.31
p > 0.04
--
--
1.29 ± 0.56
1.05 ± 0.67
p > 0.5
Weanling rats (age 30 days) were exposed to oxidant gas as indicated.
^C-a-tocopherol was injected ip, and exposure was continued for 3 more
days. Rats were killed, and the radioactivity in a-tocopherol and
Q'-tocopherol oxidation products was determined as described in Materials
and Methods. Tabulated values are the ratios of ^cr-tocopherol oxida-
tion products to l^C-or-tocopherol.
Rats were killed 96 hours after administration of ^C-a-tocopherol.
Weanling rats (age 30 days) were placed on a vitamin E-deficient diet
for 6 weeks. After 4 weeks on the diet, rats were exposed to 5 ppm
of N02 for 10 days and then injected with -^C-a-tocopherol. N02
exposure was continued, and 4 days later the rats were killed.
Standard Student's t-test; values of p < 0.05 are considered to be
statistically significant.
29
-------
in lungs or blood. These results are similar to those obtained
after 3 days of exposure to N02.
In some initial experiments, we injected 1 (aCi of 3H-L-leucine
with the l4C-a-tocopherol so as to monitor lung metabolism for
use as a baseline reference. We found that the incorporation
rate into protein was Less than 100 dpm/mg, thus providing no
advantage for ascertaining the state of the animal, relative to
metabolism of l4C-o;-tocopherol.
14C-RETINOL ACETATE METABOLISM
We examined the metabolic fate of -"-"C-retinol acetate by TLC
of lipid extracts of tissues from N02-exposed and control rats.
Figure 11 shows the results for liver. Only a single radioactive
peak at R 0.78 was found. This material did not migrate with
either retinol acetate (R 0.45) or retiriol (R 0.10),, and its
identity remains unknown. Lungs, blood, and kidney from NOS -
exposed and control rats all showed similar results: only one
peak of radioactivity at R 0.78 was observed. No significant
differences between exposed and control animals were observed
in the amounts of this material.
ISOLATION AND CHARACTERIZATION OF UDP-GLUCURONIC ACID:DIHYDRO-
a-TOCOPHERONOLACTONE GLUCURONIC ACID TRANSFERASE
We assayed UDP-GlcUA:a-tocopheronolactorie glucuronosyl transferase
activity by following the transfer of l4C-GlcUA from UDP-l4C-GlcUA
to a-tocopheronolactone to yield dihydro-o'-tocopheronolactone-
l4C-glucuronide. The latter was readily isolated by chromatography
30
-------
3000
2000
a
a
1000
EXPOSED LIVER
10 ppm NO
ORIGIN
4000
3000
2000
1000
RETINOL
ACETATE
SOLVENT
FRONT
CONTROL LIVER
1
T f
ORIGIN RETINOL
RETINOL
ACETATE
SOLVENT
FRONT
FIGURE 11 THIN-LAYER CHROMATOGRAPHY OF 14C-RETINOL ACETATE AND METABOLITES
FROM LAYERS OF NO2-EXPOSED AND CONTROL RATS
Authentic retinol acetate is indicated by the hatched lines.
31
-------
on Whatmann 3-mm paper using 95% ethanol;! M ammonium acetate,,
pH 7.5 (7:3, v/v). Under these conditions, dihydro-Q'-
tocopheronolactone-l4C-glucuronide migrated with an R of 0.79,
whereas UDP-GlcUA, GlcUA-l-P04, and GlcUA migrated with an R of
less than 0.5. Under the assay conditions described in Materials
and Methods, incorporation of """ C-GlcUA into dihydro-o/-tocophero-
nolactone- C-glucuronide was linear with time for up to 1 hour
and with enzyme protein for up to 0.7 mg per incubation (Figures
12 and 13). Glucuronosyltransferase activities are often linear
for incubation periods of up to 6 hours or longer.31 The initial
rate of the reaction was somewhat low (Figure 12), presumably be-
cause of the requirement for reduction of Q'-tocopheronolactone to
dihydro-a-tocopheronolactone before the addition of the glucuronic
acid moiety. This was indicated by the finding that maximum
100
FIGURE 12 FORMATION OF a-TOCOPHERONOLACTONE
GLUCURONIDE WITH TIME
Assays were performed using a-tocopheronolactone
as substrate as described under Materials and
Methods. Incubations contained approximately 0.5
mg of microsomal protein.
32
-------
IT
O
U
Q
O
tr.
a.
25
20
15
10
FIGURE 13
O
I
I
I
200 400 600
PROTEIN M9/0.05 ml
800
FORMATION OF a-TOCOPHERONOLACTONE
GLUCURONIDE AT VARYING PROTEIN
CONCENTRATIONS
incorporation occurred when NADH was included in the incubation
as a potential cofactor for the reduction of a-tocopheronolactone.
XADPH also stimulated the transferase reaction but at a lower
rate,, whereas FADH2 had no effect.
As shown in Figure 14; the enzyme showed typical Michaelis-Menten
kinetics using a-tocopheronolactone as substrate. A Km value
for ?<-tocopheronolactone of approximately 2.8 mM was determined
by the method of Lineweaver and Burke (Figure 14,, inset).
Substrate inhibition by a-tocopheronolactone was observed at
concentrations of 50 mM or higher. We observed maximum incor-
poration of glucuronic acid into dihydro-o'-tocopheronolactone
glucuronide at a UDP-GlcUA concentration of approximately 0.01 M.
The estimated Km value for UDP-GlcUA was 8 mM^ as shown in
Figure 15.
33
-------
10 20 30 40 50 100
a-TOCOPHERONOLACTONE CONCENTRATION
FIGURE 14 SATURATION OF GLUCURONOSYL TRANSFERASE
WITH a-TOCOPHERONOLACTONE
(a) Determination of Km for a-Tocopheronolactone
by the Method of Lineweavei-Burke
The enzyme did not require divalent cations for catalytic
J L I | L
activity. Most divalent cations tested (Co , Zu , Xi ,
1 I _| L
Mn , Ca ) inhibited enzyme activity, as indicated in Table 4,
i i_ _j |_
although Mn and Ca showed a slight stimulation at low con-
_j i_
centrations (10 mM or less). Sn stimulated the enzyme reac-
tion to the greatest extent (162% of control) but only at low
_j I
concentrations (2 mM). Higher concentrations of Sn were not
tested. These results suggest that divalent cations are not
required for glucuronosyl transferase activity, although cer-
tain ones are stimulatory. Additional work is required to
clarify this effect.
The glucuronosyl transferase activity is found predominantly in
the liver of the rat, as shown by the tissue distribution study
34
js-C
-------
20 r
15
DC
O
10
Q
§ 5
D-
5 10 15
UDP-GlcUA CONCENTRATION
20
25
FIGURE 15 SATURATION OF GLUCURONOSYL TRANSFERASE
WITH UDP-GlcUA
summarized in Table 5. Kidney has approximately 5% of the total
activity of the liver, whereas spleen, brain, and lung have less
than 17o. The activity of the enzyme in heart, fat, and serum was
below the limits of detection.
During these studies, Dr. Gustave Freeman provided three samples
of monkey (Macaca speciosa) liver from a control monkey and two
monkeys exposed to 2 and 9 ppm N02 for approximately 9 years.
We assayed these tissues for UDP-GlcUA:dihydro-a-tocopheronolactone
glucuronosyl transferase, and Figure 16 presents the results.
Enzyme activity was inversely proportional to the N02 concentration.
35
-------
Table 4. EFFECT OF DIVALENT CATIONS
ON GLUCURONOSYL TRANSFERASE ACTIVITY
Metal
++
Co
Zn^
SnH-
Ni"
++
Mg
Ca^
j i_
1 r
Mn
Cu"^
Metal concentration, mM
2
162
5
57
73
59
146
102
78
0
10
42
38
18
129
122
72
25
54
50
< 1
0
24
59
30
100
29
5
Metals were added as the dichloride salt to the
standard transferase incubation mixture described
in Materials and Methods. Tabulated values are
percentage of transferase activity relative to
control incubations lacking added metal.
36
-------
Table 5. GLUCURONOSYL TRANSFERASE ACTIVITY
IN RAT TISSUES
Tissue
Liver
Kidney
Spleen
Brain
Lung
Heart
Fat
Blood
Serum
Specific
Activity3
32.3
7.7
2.1
1.0
0.8
<0.2
<0.2
0.8
<0.2
Total Tissue
Activity*3
581
26.8
2.0
1.6
1.5
<0.3
--
--
--
One unit of enzyme activity will convert 1 umole
of substrate per minute in the standard assay used
Units x 103/g (ml) of fresh tissue.
Units x 10
whole organ.
37
-------
50 r
J
024 6 8 10
CONCENTRATION OF NO2 ppm
FIGURE 16 EFFECT OF NO2 EXPOSURE ON
GLUCURONOSYLTRANSFERASE
ACTIVITY IN THREE SAMPLES
OF MONKEY LIVER
38
-------
SECTION VI
DISCUSSION
The objective of this project was to determine whether exposure
of rats to an atmosphere containing N02 or 03 has observable
effects on the disposition and metabolism of vitamins E and A.
Both these substances are susceptible to oxidation, and they
may be oxidized in lung tissue in situ during exposure to N02
or 03 .
We have demonstrated that the uptake of 1 C-o'-tocopherol in N02 -
exposed lungs is increased by approximately 50% over that in
control lungs when measured either by uptake per milligram of
lung protein (Figure 1) or by uptake in the total lung (Figure 4),
This increase was not observed in liver and blood, the retention
of a-tocopherol being the same for both exposed and control
animals (Figures 2 and 5). N02 exposure did not affect the half-
life of l4C-a-tocopherol in lungs, liver, and blood. The -^C-a-
tocopherol was cleared from blood twice as fast as from lungs or
liver (Table 2). These data suggest that lung tissue takes up
an increased amount of the antioxidant ff-tocopherol in response
to exposure to an oxidizing atmosphere of N02.
We investigated the effects of different concentrations of N0g
on the ratio of cv-tocopherol oxidation products to Q'-tocopherol
(Table 3). The livers of animals exposed to 10, 14, and 20 ppm
39
57
-------
N02 had a significantly higher ratio of oxidation products than
those from control animals. Blood had a significantly larger
amount of oxidation products only in animals exposed to 20 ppm
N02, whereas lung tissue did not show any significant difference
in oxidation products for any exposure regime.
Because exposure to 14 and 20 ppm N02 had no effect on the amount
of oxidation products found in lung tissue, and because we wanted
to examine the effects of lower concentrations of N02, we used
two stratagems in an attempt to accentuate the appearance of
oxidation products. First we extended the initial exposure period
to 3.5 weeks in the experiment using 10 ppm N02. As before, the
lungs of the exposed rats had the same proportion of oxidized
products as the controls. Also, the livers from the exposed
rats had significantly larger amounts of: oxidation products than
the controls. This difference was similar to that found in the
14 ppm and 20 ppm exposure experiments.
The second technique was to deplete the endogenous stores of
vitamin E in rat tissue by placing weanling rats on a vitamin E-
deficient diet for 4.5 weeks before exposing them to 5 ppm NOZ ,
the lowest concentration of N02 used in these studies. Bieri"
showed that weanling rats fed a vitamin E-deficient diet for
8 weeks still grew and displayed no overt: signs oE disease. At
4.5 and 8 weeks, respectively, the following percentages of
a-tocopherol remained in various tissues: plasma, 1% and 1%;
liver, 12% and 6%; fat, 28% and 10%; heart, 33% and 20%; skeletal
muscle, 64%, and 45%; and testis, 31?0 and 28%>. Thus, from our
experiment, which terminated at 6 weeks, we can surmise that,
40
-------
when l4C-o'-tocopherol was injected after 5.5 weeks on diet,
the stores of endogenous tocopherol in liver, fat, and plasma
had been significantly depleted without pathological effects
on the animals. Although the tocopherol content of heart was
still dropping by 8 weeks, the levels in the testis and skeletal
muscle had leveled off and were declining slowly. If the tocoph-
erol content of lung tissue were influenced similarly to that
of skeletal muscle or heart, we could assume that the content
was below 50%. Under these circumstances, a reasonable expecta-
tion is that any vitamin E metabolism would engage a larger
proportion of administered l4C-Q'-tocopherol and, thus, appear
amplified.
In contrast to results from previous experiments, the results
of this experiment (Table 3) showed that the N02 exposure did
not significantly alter the level of oxidation products compared
with control values in any of the three tissues examined. Possibly,
continuous exposure to 5 ppm N02 (and perhaps to even lower con-
centrations) for a longer time would result in the appearance of
Q'-tocopherol oxidation products. However, in these experiments,
the lowest level of N02 that enhanced the detection of a-tocopherol
oxidation products was 10 ppm.
The results of exposure to 1 ppm 03 were the same as those for
exposure to 10 to 14 ppm N02. a-Tocopherol oxidation products
were elevated in the liver, but the lung and blood were unaffected.
These results are not unexpected, because 03 at 1 ppm causes
morphological and biochemical alterations of the lung that are
very similar to those produced by N02 exposure at about 14 ppm. '*
41
-------
The chemical modes of action of the two gases are different, 5
and the effects they produce, although similar, can be readily
distinguished by careful observation. For example, N02 exposure
typically causes a doubling of glucose-6-phosphate dehydrogenase
activity in lung, whereas 03 exposure increases the activity
fi o c
by only 507o. > Other biochemical differences can also be
distinguished, as can morphological differences.
During the first week of exposure, 03 stimulates the infiltration
of many more macrophages into the alveoli and of monocytes into
the interstitial spaces than does N02. N02 tends to cause strati-
fication of the nonciliated cells in the terminal bronchioles.33-'37
Long-term exposure to N02 (30 days or more) causes additional
changes not seen with 03. Proteinaceous crystalloids appear in
nonciliated cells and Later in ciliated cells.33'08 Also,
ciliated cytoplasmic vacuoles appear, basement lamina thicken,
and the diameter of collagen fibrils increases.39'40
An unexpected result was that lung tissue of N02-exposed animals
showed no significant differences in the levels of a-tocopherol
oxidation products compared with lungs of control animals,
whereas significant elevations were consistently observed in
the liver (Table 3). This is particularly surprising considering
that the exposed lung took up larger amounts of 14 C-o/-tocopherol
(Figure 4). We might expect that lung tissue being directly
exposed to the oxidizing gas N02 and having a greater retention
of l4C-o/-tocopherol--would show the most pronounced effect on
a-tocopherol metabolism. Instead, the products of cv-tocopherol
oxidation apparently are rapidly cleared from lung tissue and
42
-------
are transported to the liver where they accumulate. This process
continues for at least 4 days after administration of 1 C-o;-
tocopherol, since the level of cv-tocopherol oxidation products
present in the liver is still increasing at this time (Figure 9).
The absence of a linear relationship between the concentration
of N02 used in the exposure experiment and the level of Q'-tocopherol
oxidation products found in the liver probably results from the
relative rates of accumulation, subsequent metabolism including
conjugation with glucuronic acid, and excretion. Other factors
may also be involved such as artifactual oxidation of a-tocopherol
or nonuniform response of animals to N02 exposure. Additional,
more carefully controlled experiments are needed to delineate
the system further.
Another interesting observation is that, in the three experi-
ments involving long exposures to N02 (Table 3), the proportion
of oxidation products found in the liver and blood of the control
rats far exceeded that found in the control rats in the short-
term experiments. The lungs, on the other hand, had the same
proportion of oxidation products in all experiments. The only
major difference between the two kinds of experiments is the
age of the animals at the time of l4C-a-tocopherol injection
(Figure 10). In the short exposure experiments, the animals
were about 4.5 weeks old, whereas in the long-term exposure
experiments they were 7, 7,5, and 9.5 weeks old. A tempting
speculation is that, as the animals age, larger amounts of
a-tocopherol oxidation products are generated throughout the
body and are accumulated in the liver for subsequent disposal.
43
-------
The presence of a-tocopherol oxidation products in tissue has
been noted before. In rats fed a vitamin E-deficient diet for
several months, Peake and Bieri41 found that ITL of °H-a/-tocopherol
appeared in the liver as oxidation products 27 hours after ip
injection. Csallany et al.42 recovered only 257= of total radio-
activity in rat liver as unchanged a-tocopherol 2 days after
injection of l4C-a-tocopherol. In contrast, Krisriamurthy and
43
Bieri found that over 907o of orally administered tocopherol
was recovered in rat liver regardless of the time interval up
to 21 days after administration. Similarly, they found only 17o
of the label from orally administered l4C-o?-tocopherol to be
44
excreted in the urine, whereas Simon et al. found that up to
307o of the label from l~C-a-tocopherol administered intravenously
to rabbits was excreted in the urine. Other urinary metabolites
resulting from further side chain oxidation of o'-tocopheronic
4 e
acid have been described. Clearly, the route of administration,
the nutritional status of the animals, and the animal species
used are important variables affecting the metabolism of
^-tocopherol.
Good quantitation is difficult to obtain in vitamin E assays.
Thus, researchers must take extreme precautions to prevent
spurious oxidation of tocopherol during laboratory manipulations
because of its sensitivity to oxidation by air and by tissue-
derived heme compounds. In our work, routine precautions to
minimize artifactual oxidation of a-tocopherol included working
in a darkened room, using deaerated solvents containing anti-
oxidants such as BHT or pyrogallol, and performing all
44
-------
concentrations under an inert atmosphere of nitrogen. In spite
of these precautions, the high proportion of 1'iC-Q'-tocopherol
recovered as oxidation products (up to 56% of recovered label,,
Table 3) may indicate artifactual oxidation. Nevertheless, we
found (Table 3) that the ratios of oxidized products (quinone
plus dimer) to unchanged a-tocopherol (a-tocopherol plus origin
material) were reproducible within a single group of animals; but
between groups, including both exposed and controls, considerable
variation was observed. If this variation is artifactual it is
curious that only in the groups of older animals were the ratios
consistently higher than in the groups of younger animals.
Oxidation products were formed in all tissue examined despite
our efforts to minimize their formation by nonenzymatic arti-
factual means.
The enzymatic reactions in a-tocopherol metabolism have not
been studied previously and none of the enzymes have been
characterized. The appearance of increased levels of c^-tocopherol
oxidation products in liver due to oxidant gas exposure might
induce an elevation in levels of enzymes involved in their sub-
sequent metabolism. We chose UDP-GlcUA :o--dihydrotocopheronolactone
glucuronosyl transferase for study because it is the last enyzme
in a-tocopherol metabolism before excretion and because the
glucuronic acid donor UDP-GlcUA and the potential acceptor
o'-tocopheronolactone were readily available. This enzyme
catalyzes the transfer of glucuronic acid from UDP-GlcUA to re-
duced cv-tocopheronolactone.
45
-------
Our work demonstrates the presence of an active UDP-GlcUA:a-
tocopheronolactone glucuronosyl transferase in liver, a slight
activity in kidney, and negligible activity in the other tissues
examined. This transferase does not require divalent cations as
A C
do some glucuronosyl transferases, but it is slightly stimulated
I i_ _j i_ _i i_
by Mg , Ca , and Sn . NADH also stimulates the activity,
indicating a requirement for prior reduction of the lactone to
the dihydrolactone because only the latter can be glucuronylated.
Both activities are found in the microsomal fraction.
Liver contains UDP-glucuronic acid glucuronosyl transferase
(acceptor-unspecific) (EC 2.4.1.17), which is responsible for
the transfer of glucuronic acid from UDP-GlcUA to a variety of
phenols, alcohols, amines, and fatty acids.46 We do not know
whether the glucuronosyl transferase activity using
o'-tocopheronolactone as substrate is due to the nonspecific
glucuronosyl transferase because we did not perform substrate
competition experiments.
Long-term exposure (9 years) of monkeys to N02 resulted in a
decrease in the cellular content of transferase activity in
the liver (Figure 16). This is surprising because higher levels
of o'-tocopherol oxidation products in the liver might be expected
to produce an elevated enzyme level if any change were to occur.
Because livers from only three monkeys were analyzed we cannot
discount the possibility that the lower activities in the two
exposed livers are due to biological variation.
In animal liver, lipid-free radicals or peroxides irreversibly
oxidize a-tocopherol in small amounts to a-tocopherolquinone and
46
-------
to dimeric and trimeric metabolites. The quinone itself is
partially excreted in the feces, but most is reduced in the
liver to the hydroquinone and conjugated with glucuronic acid
and other unknown moieties before secretion in the bile and
, j- 47,48
excretion in the feces. '
Trace amounts of a-tocopherol are converted to a conjugate of
a-tocopheronic acid and excreted in the urine. This material
is presumed to arise by reduction of the quinone and conjugation
48
with glucuronic acid and other substances in kidney. Our
finding that the bulk of UDP-GlcUAia-tocopheronolactone glucuronosyl
transferase activity resides in liver, with less than 5% activity
in kidney, suggests instead that conjugationat least with
glucuronic acidoccurs in the liver. If this is the case, the
degradation of the side chain most likely also occurs in the
liver and the kidney probably serves primarily as the mechanism
for the excretion of water-soluble metabolites.
However, we must consider that the glucuronide conjugate of
ff-tocopheronic acid comprised only about 5% of the urinary
metabolites.47 Another 45% of the metabolites were also acid
hydroyzable, but these were shown not to be phosphate or sulfate
esters.47 Therefore, the possibility remains that the bulk of
the a-tocopheronic acid derivatives excreted in the urine (about
90%) may be formed and conjugated in the kidney.
In this research, we demonstrated that oxidant gas exposure
results in an increased rate of a-tocopherol oxidation. However,
the tocopherol oxidation products, principally quinone and dimer,
47
-------
are elevated only in the liver and at exposures to 10 ppm or
higher of N02 or to 1 ppm 03. Only at 20 ppm N02 did we observe
enhanced levels of oxidation products in the blood. Oxidation
products in the lungs were not elevated by any of the exposures.
The amount of a-tocopherol in the lungs of exposed animals in-
creased, whereas it remained the same in control animals.
These data suggest that lung damage caused by oxidant gas exposure
leads to an increased turnover of a-tocopherol in the lung.
Newly formed cells and cells undergoing repair require the impor-
tation of new cy-tocopherol. The a-tocopherol in damaged and
destroyed cells is released unchanged or as oxidation products
because of in situ oxidation during exposure. The released
tocopherol and tocopherol oxidation products are transported to
the liver where the oxidation products accumulate and are further
metabolized to conjugates of a-tocopheronic acid. The fate of
the dimer and trimer is unknown.
We could not demonstrate any significant difference in metabolism
of retinol acetate between N02-exposed and control animals in
the one experiment we performed. Because none of the original
l4:C-retinol acetate and only one spot of radioactive material
was recovered from any of the tissues, one possibility is that
retinol metabolism is very rapid and that tissue must be examined
sooner than 24 hours after injection. However, earlier work
indicates that retinol metabolism is not this rapid. Complete
recovery of radioactivity from 1 C-retinoic acid required 48
hours, 9 and retinyl acetate was metabolized at a nearly constant
rate for 6 to 7 days.50'51 However, these other investigators
48
-------
administered labeled vitamin A intravenously whereas we used
ip administration.
Vitamin A is susceptible to light-catalyzed isomerization and
rearrangement as well as to air oxidation. Therefore, another
possibility is that the single radioactive spot derived from
l4C-retinyl acetate is an artifact of isolation despite our
efforts to avoid this circumstance. Thus, the retinol data must
be interpreted cautiously. Additional experiments performed under
more rigorous conditions are required before we can draw meaning-
ful conclusions about the effect(s) of oxidant gases on vitamin
A metabolism.
49
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SECTION VII
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19. Stephens, R. J., M. F. Sloan, M. J. Evans, and G. Freeman.
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21. Evans, M. J., L. V. Johnson, R. J. Stephens, and G. Freeman.
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Exposure to N03 or 03. Lab. Invest. In press, 1976.
22. Nakamura, T., and S. Kijima. Studies on Tocopherol Derivatives.
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23. Jones, E.R.H., and R, M. Evans. Vitamin A Synthesis.
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25. Cheeseman, G.W.H., I. Heilbron, E.R.H. Jones, F. Sondheimer,
and B.C.L. Weeden. Studies in the Polyene Series. XXXIII.
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26. Brown, C. A. Facile Reaction of Potassium Hydride with
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Dioxide and Ozone). Arch Environ Health. 29(4):203-210,
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28. Freeman,, G., S. C. Crane, R. J. Stephens, and N. J. Furiosi.
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32. Bieri, J. G. Kinetics of Tissue a-Tocopherol Depletion and
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39. Stephens, R. J., G. Freeman, S. C. Crane, and N. J. Furiosi.
Ultrastructural Changes in the Terminal Bronchiole of the
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41. Peake, I. R., and J. G. Bieri. Alpha- and Gamma-Tocopherol
in the Rat: In Vitro and I_n Vivo Tissue Uptake and
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42. Csallany, A. S., H. H. Draper, and S. N. Shah. Conversion
of d-a-Tocopherol-ll4C to Tocopherolquinone In Vivo.
Arch Biochem Biophys. 98_(1) : 142-145, July 1962.
43. Krisnamurthy, S., and J. G. Bieri. The Absorption, Storage,
and Metabolism of a-Tocopherol-C14 in the Rat and Chicken.
J Lipid Res. 4(3):330-336, July 1963.
44. Simon, E. J., A. Eisengart, L. Sundheim, and A. T. Milhorat.
The Metabolism of Vitamin E. II. Purification and
Characterization of Urinary Metabolites of o-Tocopherol.
J Biol Chem. 22_L(2): 807-817, August 1956.
45. Watanabe, M., M. Toyoda, I. Imada, and H. Morimoto.
Ubiquinone and Related Compounds. XXVI. The Urinary
Metabolites of Phylloquinone and a-Tocopherol. Chem Pharm
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46. Dixon, M., and E. C. Webb. The Enzymes. Second Ed.
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47. Bunyan, J., J. Green, A. T. Diplock, and E. E. Edwin.
Pyridine Nucleotide-Tocopheronolactone Reductase.
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55
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48. Chow, C. K., H. H. Draper,, A. S. Csallany, and M. Chiu.
The Metabolism of C14-a-Tocopheryl Quinone and Cl4-a-
Tocopherol Hydroquinone. Lipids. _2_(5) -.390-396, September
1967.
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50. Roberts, A. B., and H. F. De Luca. The Fat-Soluble
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in the Rat. Biochem J. 102:600-605, February 1967.
56
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TECHNICAL REPOUT DATA
f rnut liiuruct'ons on ilif /t" nc before r
1. REPORT NO.
EPA-6 00/1-76-028
2.
3. RECIPIENT'S ACCESSION1 NO.
4. TITLE AND SUBTITLE
THE PHARMACODYNAMICS OF CERTAIN ENDOGENOUS MAMMALIAN
ANTIOXIDANTS DURING N02 EXPOSURE
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
August 1976
7. AUTHOR(S)
Kenneth D. Lunan and Alan E. Brandt
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Biochemistry Laboratory
Stanford Research Institute
Mcnlo Park, California 94025
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1713
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Rats exposed to atmospheres containing nitrogen dioxide (N02) in excess of
10 ppm showed a 50% increase in uptake of *4C-a-tocopherol by the lung when compare
with control rats maintained in ambient air. This increase was not observed in
liver or blood, the retention of lt+C-a-tocopherol being the same in exposed and
control animals. N02 exposure did not affect the half-life of 1L|C-a-tocopherol in
lung, liver, or blood. The liver of rats exposed to greater than 10 ppm N02 or to
1 ppm ozone showed a statistically significant (P < 0.05) increase in the level of
a-tocopherol oxidation products compared with control rat liver, as judged by an
increase in the ratio of a-tocopherol quinone plus a-tocopherol dimer to a-tocophe-
rol. This increase was limited to the liver and was not observed in either lung or
blood. Liver, lung, and blood of vitamin E-deficient rats exposed to 5 ppm N02 did
not show any statistically significant increase in a-tocopherol oxidation products
when compared with control tissues. No effect of N02 in C-retinol acetate meta-
bolism was observed. This research resulted in the first description of an enzyme
involved in a-tocopherol metabolism - namely, a UDP-glucuronic acid:dihydro-o-toco-
pheronolactone glucuronosyl transferase, the final enzyme in a-tocopherol metabolisn
before excretion. The glucuronosyl transferase is a microsomal enzyme found predom
inantly in the liver, and does not require a divalent cation for activity, althroug
it is stimulated bv Sn"*~+. Ca.++. and Mg++.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENT1FIERS/OPEN ENDED TERMS
COSATI Held/Group
Pharmacology
Biomedical measurements
nitrogen dioxide
ozone
enzymes
a-tocopherol
06, 0, F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (Tins Report)
__UNCLAaS.LElED_
21. NO. OF PAGES
68
20. SECURITY CLASS (Tins paf
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
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