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 animals—but 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

-------
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

-------
                          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

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             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

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                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

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             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

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            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

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    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 conjugation—at least with



glucuronic acid—occurs 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

-------
                           SECTION VII

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     Metabolites of Phylloquinone and a-Tocopherol.   Chem Pharm
     Bull.   22(1):176-182,  January  1974.

46.  Dixon,  M.,  and E.  C.  Webb.  The Enzymes.  Second Ed.
     New York, Academic Press Inc.,  1964.  p. 712.

47.  Bunyan,  J., J.  Green,  A. T. Diplock,  and E. E.  Edwin.
     Pyridine Nucleotide-Tocopheronolactone  Reductase.
     Biochim Biophys Acta.   49:420-422,  May  1961.
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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-
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     1967.

49.  Draper,  H. H.,  and A.  S.  Csallany.   The  Fat-Soluble Vitamins.
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     The University of Wisconsin Press,  L970.   p.  347.

50.  Roberts, A. B., and H. F. De Luca.   The Fat-Soluble
     Vitamins.  H. F. De Luca and J.  W.  Suttie,  eds.  Madison,
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51.  	•  Pathways of Retinol and Retinoic Acid Metabolism
     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)
                                            57

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