EPA-600/1-76-028 August 1976 Environmental Health Effects Research Series ------- 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. ------- 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 ------- 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 ------- 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 ------- 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 ------- 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 ------- 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 ------- 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 ------- 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 ------- 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 ------- TLC Thin-layer chromatography _| |_ Sn Tin, divalent ion UDP Uridine diphosphate V Velocity, initial v/v Volume per volume -H- Zn Zinc, divalent ion ------- 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. ------- 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. ------- 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. ------- 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. ------- 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 ------- 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 ------- 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 ------- 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. ------- 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 ------- ? "-\ 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 ------- 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 ------- 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 ------- 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 ------- SECTION VII REFERENCES 1. Evans, H. M., and K. S. Bishop. The Existence of a Hitherto Unrecognized Dietary Factor Essential for Reproduction. Science. .56(1458):650-651, December 1922. 2. . On the Existence of a Hitherto Unknown Dietary Factor Essential for Reproduction. Am J. Physiol. 63(3):396- 397, February 1923. 3. Gross, S., and D. K. Melhorn. Vitamin E, Red Cell Lipids and Red Cell Stability in Prematurity. Ann NY Acad Sci. .203:141-162, December 1972. 4. Fletcher, B. L., and A. L. Tappel. Protective Effects of Dietary a-Tocopherol in Rats Exposed to Toxic Levels of Ozone and Nitrogen Dioxide. Environ Res. 6_(2) : 165-175, June 1973. 5. Green, J. Vitamin E and the Biological Antioxidant Theory. Ann NY Acad Sci. 203:29-44, December 1972. 6. Chow, C. K., and A. L. Tappel. An Enzymatic Protective Mechanism Against Lipid Peroxidation Damage to Lungs of Ozone-Exposed Rats. Lipids. T_:5].%-52k, August 1972. 7. Nair, P. P. Vitamin E and Metabolic Regulation. Ann NY Acad Sci. 203:53-61, December 1972. 8. McCay, P. B., P. M. Pfeifer, and W. H. Stife. Vitamin E Protection of Membrane Lipids During Electron Transport Functions. Ann NY Acad Sci. .203:62-73, December 1972. 9. Carpenter, M. P. Vitamin E and Microsomal Drug Hydroxylations. Ann NY Acad Sci. .203:81-92, December 1972. 51 ------- 10. Stumpf, P. K. Metabolism of Fatty Acids. Ann Rev Biochem. J38:159-212, 1969. 11. Hopkins, F. G. Feeding Experiments Illustrating the Importance of Accessory Factors in Normal Diets. J Physiol. 44(5,6) = 425-460,, July 1912. 12. von Euler, H., P. Karrer, and M. Rydbom. Uber die Beziehungen zwischen A-Vitaminen und Carotinoiden. Chem Ber. 62(6):2445- 2451,, June 1929. 13. Rosso, G. C., L. De Luca, C. D. Warren, and G. Wolf. Enzymatic Synthesis of Mannosyl Retinyl Phosphate from Retinyl Phosphate and Guanosine Diphosphate Mannose. J Lipid Res. 6(3):235-243, May 1975. 14. Edwin, E. E., A. T. Diplock, J. Bunyan, and J. Green. Studies on Vitamin E VI: The Distribution of Vitamin E in the Rat and the Effect of a-Tocopherol and Dietary Selenium on Ubiquinone and Ubichromenol in Tissues. Biochem J. 7JJ( 1): 91-105, April 1961. 15. Freeman, G., and G. B. Haydon. Emphysema After Low-Level Exposure to N02. Arch Environ Health. 8(1):125-128, January 1964. 16. Freeman, G., S. C, Crane, N. J. Furiosi, R. J. Stephens, M. J. Evans, and W. D. Moore. Covert Reduction in Ventilatory Surface in Rats During Prolonged Exposure to Subacute Nitrogen Dioxide. Am Rev Resp Dis. 106(4):563-579, October 1972. 17. Freeman, G., L. T. Juhos, N. J. Furiosi, R. Mussenden, R. J. Stephens, and M. J. Evans. Pathology of Pulmonary Disease from Exposure to Interdependent Ambient Gases (Nitrogen Dioxide and Ozone). Arch Environ Health. 2£(4):203-210, October 1974. 18. Stephens, R. J., G. Freeman, and M. J. Evans. Early Response of Lungs to Low Levels of Nitrogen Dioxide. Arch Environ Health, 24(3):160-179, March 1972. 52 ------- 19. Stephens, R. J., M. F. Sloan, M. J. Evans, and G. Freeman. Early Response of Lung to Low Levels of Ozone. Am J Pathol. _74(l):31-57, January 1974. 20. Evans, M. J., L. J. Cabral, R. J. Stephens, and G. Freeman. Transformation of Alveolar Type 2 Cells to Type 1 Cells Following Exposure to N02. Exp Mol Pathol. 22(1):142-150, February 1975. 21. Evans, M. J., L. V. Johnson, R. J. Stephens, and G. Freeman. Renewal of the Terminal Bronchiolar Epithelium Following Exposure to N03 or 03. Lab. Invest. In press, 1976. 22. Nakamura, T., and S. Kijima. Studies on Tocopherol Derivatives. IV. Hydroxymethylation Reaction of (3, y'^ocopherol and Their Model Compounds with Boric Acid. Chem Pharm Bull. 20(8): 1681-1686, August 1972. 23. Jones, E.R.H., and R, M. Evans. Vitamin A Synthesis. British Patent 696,235 (to Glaxo Laboratories, Ltd.) August 26, 1953. Chem Abstr. .50, 406i, August 1953. 24. Attenburrow, J., A.F.B. Cameron, J. H. Chapman, R. M. Evans, B. A. Hems, A.B.A. Jansen, and I. Walker. A Synthesis of Vitamin A from Cyclohexanone. J Chem Soc. pp. 1094-1111, March 1952. 25. Cheeseman, G.W.H., I. Heilbron, E.R.H. Jones, F. Sondheimer, and B.C.L. Weeden. Studies in the Polyene Series. XXXIII. The Preparation of 6-Methylocta-3:5:7-trien-2-one, a Key Intermediate for the Synthesis of Vitamin A and Its Analogues. J Chem Soc. pp. 2031-2035, August 1949. 26. Brown, C. A. Facile Reaction of Potassium Hydride with Ketones. Rapid Quantitative Formation of Potassium Enolates from Ketones via Kaliaton. J Org Chem. _39(9) : 1324-1325, May 1974. 27. Freeman, G., L. T. Juhos, N. J. Furiosi, R. Mussenden, R. J. Stephens, and M. J. Evans. Pathology of Pulmonary Disease from Exposure to Interdependent Ambient Gases (Nitrogen Dioxide and Ozone). Arch Environ Health. 29(4):203-210, October 1974. 53 ------- 28. Freeman,, G., S. C. Crane, R. J. Stephens, and N. J. Furiosi. Pathogenesis of the Nitrogen Dioxide-Induced Lesion in the Rat Lung: A Review and Presentation of New Observations. Amer Rev Respir Dis. _98(3):429-443, September 1968. 29. Grahm, A. B., and G. C. Wood. The Phospholipid-Dependence of UDP-Glucuronosyltransferase. Biochem Biophys Res Commun. 37(4):567-575, November 1969. 30. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein Measurement with the Folin Phenol Reagent. J Biol Chem. 193(1):265-275, November 1951. 31. Brandt, A. E., J. Distler, and G. W. Jourdian. Biosynthesis of the Chondroitin Sulfate-Protein Linkage Region: Purifica- tion and Properties of a Glucuronosyltransferase from Embry- onic Chick Brain. Proc. Nat. Acad. Sci. .64(1), 374-380 (1969). 32. Bieri, J. G. Kinetics of Tissue a-Tocopherol Depletion and Repletion. Ann NY Acad Sci. 203:181-191, December 1972. 33. Stephens, R. J., and G. Freeman. Unpublished data. 34. Stephens, R. J., G. Freeman, and M. J. Evans. Early Re- sponse of Lungs to Low Levels of Nitrogen Dioxide. Arch Environ Health. 24:160-179, March 1972. 35. Roehm, J. N., and D. B. Menzel. Antioxidants Vs. Lung Disease. Arch Int Med Symp. 9_:228~233, March 1971. 36. Lunan, K. D., R. J. Stephens, and P. Short. Manuscript in preparation. 37. Chow, C. K., C. J. Dillard, and A. L. Tappel. Glutathione Peroxidase System and Lysozyme in Rats Exposed to Ozone or Nitrogen Dioxide. Environ Res. 7/3):311-319, June 1974. 38. Stephens, R. J., M. F. Sloan, M. J. Evans, and G. Freeman. Early Response of Lung to Low Levels of Ozone. Am J Pathol. 74:31-57, January 1974. 54 ------- 39. Stephens, R. J., G. Freeman, S. C. Crane, and N. J. Furiosi. Ultrastructural Changes in the Terminal Bronchiole of the Rat During Continuous,, Low-Level Exposure to Nitrogen Dioxide. Exp Mol Pathol. _14_(1):1-19, February 1971. 40. Stephens, R. J., G. Freeman, and M. J. Evans. Ultrastructural Changes in Connective Tissue in Lungs of Rats Exposed to N02. Arch Int Med Symp. ^: 80-90, March 1971. 41. Peake, I. R., and J. G. Bieri. Alpha- and Gamma-Tocopherol in the Rat: In Vitro and I_n Vivo Tissue Uptake and Metabolism. J Nutr. 101:1615-1622, December 1971. 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 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. 55 ------- 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. 49. Draper, H. H., and A. S. Csallany. The Fat-Soluble Vitamins. H. F. De Luca and J. W. Suttie, eds. Madison, Wisconsin, 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, Wisconsin, The University of Wisconsin Press, 1970. p. 227. 51. Pathways of Retinol and Retinoic Acid Metabolism in the Rat. Biochem J. 102:600-605, February 1967. 56 ------- 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 ------- |