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Mainstream and sidestream cigarette smoke exposure increases retinol in guinea pig lungs
Free Radical Biology & Medicine, Vol. 18, No. 3, pp. 507-514, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00
Pergamon
0891-5849(94)00161-8
Original Contribution MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE EXPOSURE INCREASES RETINOL IN GUINEA PIG LUNGS
SHYAMALI M U K H E R J E E , * T U L T U L N A Y Y A R , * F R A N K CHYTIL, t and SAL1L K . D A S * Departments of Biochemistry, *Meharry Medical College and Wanderbilt University, Nashville, TN, USA
(Received 7 April 1994; Revised 27 June 1994; Accepted 13 July 1994) A b s t r a c t - - W e have studied in guinea pigs the effects of cigarette smoke exposure on vitamin A (retinol) levels in plasma, lung, lung lavage, and liver. Smoke was generated from 1R3F cigarettes in a smoke exposure instrument designed by University of Kentucky Tobacco and Health Research Institute. Three-week-old male guinea pigs were exposed to mainstream, sidestream, or sham smoke, generated twice daily from three cigarettes for 6 weeks. In addition, some animals were kept as room controls for some time. After 6 weeks of smoke exposure, some animals were allowed to recover for 6 weeks without smoke. After 6 weeks of smoking, the plasma retinol levels were lower in both smoke exposed groups when compared to the values in the sham group. Furthermore, in comparison to the sham group, the mainstream and sidestream smoke exposed groups showed a 7.6- and 8.3-fold increase in the levels of lung retinol, respectively. After the 6-week recovery period, plasma retinol of both smoke-exposed groups reached the control levels. In contrast, withdrawal of smoking did not show such an effect on the lung retinol level in both mainstream or sidestream groups. Electronmicroscopy of the lungs showed deleterious alterations in the morphology of the lungs in both mainstream and sidestream groups. Although the mechanism(s) involved in the elevation of retinol content of the lung due to smoke exposure remains to be elucidated, it is of interest that elevation of retinol content and alteration of lung morphology occurred not only in the mainstream smoke exposed but also in the sidestream group. K e y w o r d s - - S m o k i n g , Mainstream, Sidestream, Lung, Vitamin A, Guinea pig, Free radicals
lung. On the other hand, it is well documented that vitamin A (retinol) and its derivatives are involved in the differentiation and maintenance of the proper differentiated state of the lungs. 9 The objective of this research was to determine whether or not mainstream and sidestream cigarette smoke exposure causes modulation in the retinol content of plasma, lung, lung lavage, and liver. As some metabolic characteristics in guinea pigs, such as, for example, those of lung surfactant development, are more similar to humans than in other experimental animals, 1° guinea pigs were used as animal model.
INTRODUCTION
Cigarette smoking has been implicated in the pathogenesis of various types of pulmonary diseases. 1-3 In addition, a recent study indicated that nonsmokers who are exposed to smoke in the household suffer from alterations in lung function. 4 Recent studies also suggest that there is a small but consistent increased risk of lung cancer from passive smoke exposure. 5 We have recently reported that cigarette smoke exposure causes an increase in the free radical-mediated lipid peroxidation potential of erythrocytes in guinea pigs under both mainstream and sidestream conditions. 6 It is possible that the increase in lipid peroxidation of erythrocytes in smoke-exposed animals is associated with the inability of the erythrocytes to quench the free radicals because of deficiency of some antioxidants. In fact, lower levels of carotene and vitamin C have been reported in plasma of smokers. 7'8 Currently, it is not known whether cigarette smoke exposure causes alterations in the vitamin A status in
MATERIALS AND METHODS
Animals
Male guinea pigs (Hartley strain), 3 weeks of age, weighing 2 7 5 - 3 0 0 g were obtained from C a m m Research Laboratories, New Jersey. Animals were divided into four groups: Group RC maintained as room control, Group SH maintained as sham control, Group MS maintained as mainstream smoke-exposed, and Group SS maintained as sidestream smoke-exposed. After an acclimatization period of 7 days, Group RC
Address correspondence to: Salil K. Das, Department of Biochemistry, Meharry Medical College, Nashville, TN 37208, USA.
507
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S. MUKHERJEE et al.
Table 1. Effects of Mainstream and Sidestream Cigarette Smoke Exposure on Total Particulate Matter (TPM) Inhaled, Blood Carboxyhemoglobin(COHb) Levels, Food Consumption, Body Weight Gain, and Lung Weight of Guinea Pigs Parameters
RC
SH
MS
SS
1.5 (0.2) 313 (17) 34.2 (0.8) 0.77 (0.1)
420 (10) 10.0 (0.3) 189 (16) 33.5 (1.2) 1.29 (0.2)
275 (7) 10.5 (0.2) 158 (18) 33.7 (1.1) 1.36 (0.3)
TPM (mg) COHb (%) Body weight gain (g) Daily food consumption (g) Lung weight (g/100 g body wt.)
317 (9) 35.0 (1.2) 0.79 (0.1)
RC, SH, MS, and SS groups had 3, 8, 8, and 8 animals, respectively. Values are mean and the number in parentheses represents standard error of the mean. Animals were exposed to smoke from 3 cigarettes twice daily for 6 weeks.
contained 3 animals, and other groups contained 13 animals each. The animals were housed in individual stainless steel cages in a temperature-controlled (26°C) 12/12 h light/dark cycle room. They were fed a complete life-cycle diet (Purina Diet #5025) prepared by Purina Mills, Inc., St. Louis, MO. The diet contained 35.9 ppm of carotene, 30 IU/gm of vitamin A acetate, and 50 IU/gm of vitamin E. The animals were allowed to eat and drink ad lib and were weighed at 7-day intervals. Food consumption was recorded daily.
Cigarette smoke exposure conditions Animals of MS and SS groups were exposed twice daily for 6 weeks to either MS or SS smoke from three 1R3F cigarettes in a University of Kentucky Tobacco and Health Research Institute M S - S S smoke exposure system. 6 Briefly, the smoke was generated once each minute by a 2-s 35-ml puff, and drawn into a recycle reservoir for dilution with air before distribution to the exposure chambers. Under these conditions, the animals received smoke for the first 1 6 - 2 0 s followed by air until the next puff was generated 60 s later. Thus, the animals were exposed to fresh cigarette smoke intermittently in a fashion analogous to human smoking. One advantage of this exposure system is that the tendency of animals to withhold breathing during exposures is minimized. The pumpheads of this instrument generate a constant evenpitched sound during exposures instead of the loud clicking sound of the solenoid valve that precedes the generation of puff in some other smoking machines and alerts the animals of smoke influx into the chambers. ~ Additionally, a 7-day break-in period was used in the protocol. During this period, the concentration of smoke in the puff and the number of puffs given during each exposure session were gradually increased. This
acclimatizes the animals and reduces the initial mortality due to smoke. It also discourages the animals from learning to withhold breath during smoke exposures. After acclimatization, the animals were exposed to smoke as described earlier. The cigarettes were smoked to 30.0 mm butt length. Under these conditions, 1R3F cigarettes deliver 18.1 mg of total particulate matter, 1.16 mg of nicotine, and 17.2 mg of CO per cigarette, and average about 9.5 puffs/cigarette. 6 The sham controls received a treatment identical to smoke-exposed groups but in the absence of cigarette smoke to generate similar stress conditions. After 6 weeks of smoke exposure, 5 animals from each of the SH, MS, and SS groups were maintained for an additional 6 weeks without any treatment.
Analysis of indicators of smoke exposure Exposure of animals to cigarette smoke was ascertained by measuring total particulate matter (TPM) inhaled and blood carboxyhemoglobin levels. Estimates of the daily inhaled total and cumulative particulate matter intake were made as described by Griffith and Hancock. 12Briefly, TPM was determined by extracting the filters with 100 ml of acetone, determining the absorbance at 390 nm, and multiplying the results by 100. Control values, determined before smoke exposure, permitted an evaluation of smoking machine performance on a daily basis and for the entire experiment. The difference in control TPM values and the TPM values after exposure gave an estimate of the total TPM dose received by the animals. Carboxyhemoglobin values were obtained on blood samples from the saphenous vein in a hind leg. Twenty microliter blood samples were collected, and the percent of carboxyhemoglobin was determined by the spectrophotometric procedure described by Griffith and Standaferfl 3
Smoking and lung vitamin A
509
Fig. 1. Transmission electron micrographof lung from a guinea pig exposed to mainstream smoke (3 cigarettes twice daily) for 6 weeks, showing abnormal type II cells protruded into the alveolar space (original magnification × 3400; print magnification X 2.9).
Ultrastructural analysis
Biochemical analysis
Lung samples were fixed with 2.5% glutaraldehyde in 0.1 M Sorensen's phosphate buffer, pH 7.2 for 4 h at 5°C. Tissue samples were washed for 1 h in buffer and treated for 1 h with 1% osmium tetroxide in 0.1 M Sorensen's phosphate buffer. The samples were then dehydrated in 50%, 70%, 95%, and 100% ethanol, exchanged with propylene oxide and allowed to infiltrate overnight in 1:1 propylene oxide-Epon 812 mixture. The tissue was embedded in Epon 812 according to Luft. 14 Thick sections (1 #m) were cut with a glass knife, stained with toluidine blue, and surveyed by light microscopy. For ultrastructural analysis, these sections were then stained with uranyl acetate and lead citrate. A Phillips EM 301 transmission microscope was used for electron microscopic examination.
At the end of the experiment, plasma, lungs, livers, and lung lavages were collected. Lung lavage was performed as described by us earlier. 15 In plasma and lavage samples, retinol was determined by the spectrofluorometric method of Thompson et al. 16Total retinol in the livers and lungs was assessed by the same method after alkaline hydrolysis. ~6 Thus, the values represent a sum of free retinol and retinol present in the form of retinyl esters in these tissues.
Statistical analysis Data were analyzed statistically using the Student's t test. ~7 Data are presented as mean __ standard error. Differences at p < 0.05 were considered significant.
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Fig. 2. Transmission electron micrograph of lung from a guinea pig exposed to sidestream smoke (3 cigarettes twice daily) for 6 weeks, showing abnormal type II cells protruded into the alveolar space (original magnification × 3200; print magnification x 2.8).
RESULTS
Data on total particulate matter inhaled by the animals exposed to MS and SS cigarette smoke for 6 weeks, and the corresponding values on blood carboxyhemoglobin levels, food consumption, body weights, and lung weights are shown in Table 1. Over the 6-week period, each animal from the mainstream and sidestream groups inhaled about 420 _+ 10 and 275 _+ 7 mg total particulate matter, respectively. Cigarette smoke exposure caused a 7-fold increase in the carboxyhemoglobin levels of blood in both smoke-exposed groups in comparison to the sham group. There was no significant difference between mainstream and sidestream groups. When compared to the room control and sham groups, the animals of the mainstream- and sidestream-
smoke exposed groups showed 40% to 50% less body weight gain after 6 weeks. There was no significant difference in body weight gain between the room control and sham groups. In addition, no significant difference in food consumption was noted between the experimental groups. Therefore, the depression in body weight gain in the smoke-exposed groups was not due to loss of appetite. After 6 weeks of smoke exposure, the lung weight of both smoke-exposed groups was significantly higher than that of either room control or sham control group. However, after 6 weeks of recovery, there was no significant difference in this parameter between the different experimental groups. For example, the lung weight was 0.45 _+ 0.08, 0.51 +_ 0.09, 0.57 _+ 0.1, and 0.54 _+ 0.09 g/100 g body weight, in animals of the RC, SH, MS, and SS groups, respectively. Thus the gain of lung weight in the smoke
Smoking and lung vitamin A
511
Fig. 3. Transmission electron micrograph of lung from a room control guinea pig, showing no protrusion of type II cells into the alveolar space (original magnification x 4,300; print magnification x 2.8). exposed MS or SS group was caused by the smoke and the recovery period was long enough for recovery. Ultrastructural analysis indicated that mainstream smoke exposure caused a massive accumulation of dense, amorphous granulated materials in the alveolar space (see Fig. 1) showing lung injury. Furthermore, irregular expansion of alveolar spaces and release of enlarged type II cells in the alveolar spaces was observed (see Fig. 1). In the sidestream smoke-exposed animals, no accumulation of dense, amorphous granulated material was observed in the alveolar space. On the other hand, abnormally enlarged type 1I cells were protruded into alveolar space similar to those seen in the mainstream smoke-exposed group (Fig. 2). Figure 3 shows the lung morphology of the room control. No protrusion of type II cells was observed in the alveolar space of room control animals. It should be noted that even after 6 weeks of smoke withdrawal, enlarged type II cells were released into the alveolar space of both MS- and SS-exposed groups (see Fig. 4).
Data on the effect of smoke on retinol concentration of plasma, liver, lung, and lung lavage are shown in Table 2. Several alterations were noted. In comparison to the sham group, both the mainstream and sidestream groups showed increase in the levels of retinol in the lungs, 7.6- and 8.3-fold, respectively, after the 6 weeks of smoke exposure. In addition, plasma retinol levels were reduced in both mainstream- and sidestreamsmoke exposed groups. The decrease was higher in the sidestream group (33%) than in the mainstream group (15%). Following 6 weeks of the recovery period, there was no difference in the plasma vitamin A levels between the experimental groups; however, the concentration of vitamin A in lung was still significantly higher in both MS- and SS-groups than that in the sham group. It should further be noted that the sidestream group had about 24% decrease in vitamin A concentration in the lung since the time of withdrawal from smoke exposure. No difference in the vitamin A level in liver and
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Fig. 4. Transmission electron micrograph of lung from a guinea pig exposed to mainstream smoke (3 cigarettes twice daily) for 6 weeks and maintainedfor an additional 6 weeks without any smoke exposure (original magnification× 3200; print magnification X 2.9).
lung between the control, and sham groups was found; however, the plasma vitamin A level was slightly lower in sham group than that of the room control group. The effect in the sham group may be associated with stress, because it is known that stress causes a decrease in the level of retinol in serumJ 8 No significant difference in the vitamin A level in liver, and lung lavage was found between the treatment groups. DISCUSSION Healthy lungs are equipped with structural and nonstructural defense systems to counteract the effects of a variety of potentially injurious agents present in the inspired air. 19 Disease or nutritional deficiency can alter the structural defenses and set up vicious cycles of initial lung injury. The nonstructural, or biochemical,
defense system includes superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and reduced glutathione, which protect the lung from toxic oxygen species and reactive metabolites generated by the pulmonary metabolism of a variety of foreign compounds occurring in the inhaled air. z° Singlet molecular oxygen has been shown to be generated in biological system and is capable of damaging proteins, lipids and DNA. 21 A recent epidemiological study indicates that cigarette smoking is a contributing factor for cancer of the lung. 22 Lung cancer, caused by cigarette smoking, is occurring in all race and sex groups in America. If Americans stopped smoking, 87% of lung cancer deaths could be prevented. 22 Vitamin A plays an essential role in the respiratory tract by influencing differentiation and integrity of the
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Table 2. Effects of Mainstream and Sidestream Cigarette Smoke Exposure on the Vitamin A Levels in Plasma, Liver, Lung, and Lung Lavage of Guinea Pigs Exposure Condition 6Weeks Exposure Samples Plasma (/zg/100 ml) Lung (/~g/g)
RC
SH
MS
SS
SH
MS
SS
67.8 (1.4) 0.5 (0.0)
50.5 (1.1) 0.4 (0.0) 112.5 (4.3) 130.5 (3.2)
42.9 (1.2) 3.1 (0.1) lOl.O (4.6) 124.8 (4.8)
34.0 (2.6) 3.4 (0.2) 108.6 (4.7) 117.0 (4.2)
46.4 (2.8) 0.4 (0.0) 122.9 (5.2) 104.3 (6.7)
50.9 (1.4) 2.6 (0.1) 128.1 (4.8) 123.6 (2.4)
48.2 (1.6) 2.2 (0.1) 117.7 (4.9) 96.4 (8.5)
Lung lavage (#g/mg prot.) Liver (#g/g)
Recovery Period
126.5 (3.6)
RC, SH, MS, and SS groups had 3, 13, 13, and 13 animals, respectively. Out of 13 from each of the SH, MS, and SS groups, 8 animals were sacrificed after 6 weeks of smoke exposure from 3 cigarettes twice daily; remaining 5 animals from each group were maintained for an additional 6 weeks without any treatment. Values are mean, and the number in parenthesis represents standard error of the mean. Following 6 weeks of exposure, the vitamin A levels were significantly higher in lungs and significantly lower in plasma of both MS and SS groups than those of the sham group (p < 0.05). Following 6 weeks of the recovery period, there was no difference in the plasma vitamin A levels between the experimental groups; however, the concentration of vitamin A in lung was still significantly higher in both MS- and SS-groups than that in the sham group (p < 0.0l). Lung and liver values represent total retinol recovered after alkaline hydrolysis of the tissues.
epithelial cells. 9 A d v a n c e d vitamin A deficiency results in replacement o f mucus secreting ciliated epithelium by squamous epithelium in the respiratory tract. 23 Epidemiologic studies have demonstrated an inverse relationship between vitamin A intake and lung cancer rate. a4 Our laboratory previously reported that vitamin A deficiency causes a significant decrease in superoxide dismutase, glutathione peroxidase, and reduced glutathione levels in guinea pig lung.19 Simultaneously, it causes a marked increase in microsomal oxidation. This indicates that vitamin A plays an essential role in defense mechanism o f the cells' removal o f these toxic radicals. 19 W e have also observed that smoking causes a significant modulation o f oxygen defense mechanisms o f guinea pig lung (unpublished observation). This suggests that the lung is capable o f assimilating vitamin A during oxidant exposure providing vitamin A is available in body stores or in the diet. Ultrastructural analysis in our study indicated evidence o f lung injury in guinea pigs exposed to cigarette smoke. Oxidants, such as hydrogen peroxide, present in the cigarette smoke and those produced by the macrophages stimulated by the smoke exposure are believed to play a major role in lung injury, such as chronic bronchitis, emphysema, and inflammatory diseases. 25 Substantial cell proliferation as a response to toxic lung damage is a c o m m o n p h e n o m e n o n in lung pathology. It is now well established that damage and death o f the cells lining most o f the alveolar surface, the type I alveolar cells, m a y be quickly followed by a proliferation o f type II alveolar cells. 26 Retinoids contribute to the maintenance o f the structural integrity
o f respiratory mucosa by controlling proliferation and differentiation o f epithelial cells. 27 Hence, one could expect that there will be change in vitamin A level between normal and toxic lung. In addition, carotenoids and tocopherols can protect tissues against the deleterious effects o f lipid peroxidation by singlet oxygen with their quenching abilities. 21 The chain-breaking reactions o f these antioxidants with lipid peroxyl radicals are generally regarded as being the most important. There is strong evidence that the provitamin A, beta-carotene, is more effective in this.protective effect than vitamin A itself, a4 Our study indicates that the lung o f the guinea pig exposed to both MS and SS cigarette smoke had markedly higher levels o f vitamin A than that of sham and r o o m control groups. One may speculate that the loss of vitamin A from plasma and concomitant increase in its level in the lungs m a y be due to some adaptive mechanism whereby plasma vitamin A is mobilized into the lung. Interestingly enough, it has been reported that smoke exposure causes an increase in the level of vitamin E, another fat soluble vitamin in the lungs. 28 Thus, it is possible that when intake o f vitamin A is adequate, as is the case o f this study, vitamin A m a y minimize or lessen further oxidative damage and/or stimulate cell differentiation which is critical in repairing lung tissue. However, the mechanism how vitamin A is mobilized to the lungs from other parts o f the body remains to be elucidated. Results obtained from this study indicate that sidestream smoke exposure and mainstream smoke exposure have similar effects as far as the recruitment o f
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S. MUKHERJEE et al.
vitamin A in lung is concerned. We have established earlier that both mainstream and sidestream smoke exposures cause an increase in the production of free radicals in guinea pig erythrocytes.6 Here again we found that the sidestream animals were affected. It appears that the gaseous phase of the smoke, which gives rise in carboxyhemoglobin levels, is mainly responsible for the metabolic reactions rather than the particulate matter, because the animals exposed to the sidestream smoke inhaled 35% less particulate matters than those exposed to the mainstream smoke. Consequently, not only smokers but also nonsmokers exposed passively to cigarette smoke could benefit by adjustment of their vitamin A intake. In conclusion, this study indicates that the vitamin A nutritional status may be a very important determinant of the deleterious effects of cigarette smoke in both smokers and nonsmokers exposed to passive smoke.
9. 10. 11. 12. 13. 14. 15. 16. 17.
Acknowledgements - - This work was supported by grants from
18.
the U.S. Environmental Protection Agency (R-815617), U.S. Public Health Service (HL-14214), and U.S. Agency for International Development (936-5053). The authors are grateful to Mark O. Hunt for excellent technical assistance and Ms. Susmita Chakraborty for proofreading.
19. 20.
REFERENCES
1. Auerbach, O.; Hammond, E. L.; Garfinker, L.; Benante, C. Relation of smoking and age to emphysema. Whole-lung section study. N. Engl. J. Med. 286:853-857; 1972. 2. U.S. Public Health Service (USPHS). Smoking and health: A report of the Surgeon General, DHEW Publication (PHS) 7950066. Washington, DC: U.S. Department of Health, Education, and Welfare, Public Health Service; 1979:10-35. 3. Hunninghake, G. W.; Crystal, R. G. Cigarette smoking and lung destruction. Am. Rev. Respir. Dis. 128:833-838; 1983. 4. Willers, S.; Attewell, R.; Bensryd, I.; Schutz, A.; Skarping, A.; Vahter, M. Exposure to environmental tobacco smoke in the household and urinary cotinine excretion, heavy metals retention and lung function. Arch. Environ. Health. 47:357-363; 1992. 5. Brownson, R. C.; Alavanja, M. C.; Hock, E. T.; Loy, T. S. Passive smoking and lung cancer in nonsmoking women. Am. J. Public Health 82:1525-1530; 1992. 6. Mukherjee, S.; Woods, L.; Weston, Z.; Williams, A. B.; Das, S. K. The effect of mainstream and sidestream cigarette smoke exposure on oxygen defense mechanisms of guinea pig erythrocytes. J. Biochem. Toxicol. 8:119-125; 1993. 7. Pelletier, O. Vitamin C and cigarette smokers. Ann. N. Y. Acad. Sci. 258:156-158; 1975. 8. Chow, C. K.; Thacker, R.; Changchit, C.; Bridges, R. B.; Rehm, S. R.; Humble, J.; Turbek, J. Lower levels of vitamin C and
21.
22. 23.
24.
25.
26. 27. 28.
carotenes in plasma of cigarette smokers. J. Am. Coll. Nutr. 5:305-312; 1986. Chytil, F. The lungs and vitamin A. Am. J. Physiol. 262:(Lung Cell. Mol. Physiol. 6):L517-L527; 1992. Stith, I. E.; Das, S. K. Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes. Biochim. Biophys. Acta 714:250-256; 1982. Gairola, C. G. Free lung cell response of mice and rats to mainstream cigarette smoke exposure. Toxicol. & Appl. Pharmacol. 84:567-575; 1986. Griffith, R. B.; Hancock, R. Simultaneous mainstream-sidestream smoke exposure systems. I. Equipment and procedures. Toxicology 34:123-138; 1985. Griffith, R. B.; Standafer, S. Simultaneous mainstream-sidestream smoke exposure systems. II. The rat exposure system. Toxicology 35:13-24; 1985. Luft, J. H. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414; 1961. Khan, A. Q.; Sikpi, M. O.; Das, S. K. Study of phospholipids of guinea pig lung lavage. Lipids 20:7-10; 1985. Thompson, J. N.; Erdody, P.; Brian, R.; Murray, T. K. l;quorometric determinations of vitamin A in human blood and liver. Biochem. Med. 5:67-89; 1971. Steel, R. G. D.; Torrie, J. H. Principles and procedures of statistics: A biometric approach, 2nd ed. New York: McGraw-Hill Book Company; 1980; 86-121. Takase, S.; Goda, T.; Yokogoshi, H.; Hoshi, T. Changes in vitamin A status following prolonged immobilization (stimulated weightlessness). Life Sci. 51:1459-1466; 1992. Nair, C. R.; Davis, M. M.; Das, S. K. Effect of vitamin A deficiency on pulmonary defense systems of guinea pig lung. Int. J. Vit. Nutr. Res. 58:375-380; 1988. Jenkinson, S. G.; Lawrence, R. D.; Bark, R. F.; Gregory, P. E. Non-selenium-dependent glutathione peroxidase activity in rat lung: Association with lung glutathione S-transferase activity and the effects of hyperoxia. Toxicol. & Appl. Pharmacol. 68:399-404; 1983. Mascio, P. D.; Devassagayam, T. P. A.; Kaiser, S.; Sies, H. Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers. Biochem. Soc. Trans. 18:1054-1056; 1990. Cresanta, J. L. Epidemiology of cancer in the United States. Prim. Care 19:419-441; 1992. Boren, H. G.; Pauley, J.; Wright, E. C.; Kaufmann, D. G.; Smith, J. M.; Harris, C. Cell populations in the hamster tracheal epithelium in relation to vitamin A status. Int. J. Vit. Nutr. Res. 44:382-390; 1974. Goodman, G. E.; Omenn, G. S. Carotene and retinol efficacy trail in heavy cigarette smokers and asbestos-exposed smokers. CARET coinvestigators and staff. Adv. Exp. Med. Biol. 320:137-140; 1992. Baughman, R. P.; Corser, B. C.; Strohofer, S.; Hendricks, D. Spontaneous hydrogen peroxide release from alveolar macrophages of some cigarette smokers. J. Lab. Clin. Med. 107:233237; 1986. Witschi, H.; Cote, M. G. Biochemical pathology of lung damage produced by chemicals. Fed. Proc. 35:89-94; 1976. Jetten, A. M.; Smits, H. Regulation of differentiation of tracheal epithelial cells by retinoids. Ciba FoundSymp 113:61-76; 1985. Airriess, G. R.; Changchit, C.; Chen, L. C.; Chow, C. K. Increased vitamin E levels in the lungs of guinea pigs exposed to mainstream or sidestream smoke. Nutr. Res. 8:653-661; 1988.
Pergamon
0891-5849(94)00161-8
Original Contribution MAINSTREAM AND SIDESTREAM CIGARETTE SMOKE EXPOSURE INCREASES RETINOL IN GUINEA PIG LUNGS
SHYAMALI M U K H E R J E E , * T U L T U L N A Y Y A R , * F R A N K CHYTIL, t and SAL1L K . D A S * Departments of Biochemistry, *Meharry Medical College and Wanderbilt University, Nashville, TN, USA
(Received 7 April 1994; Revised 27 June 1994; Accepted 13 July 1994) A b s t r a c t - - W e have studied in guinea pigs the effects of cigarette smoke exposure on vitamin A (retinol) levels in plasma, lung, lung lavage, and liver. Smoke was generated from 1R3F cigarettes in a smoke exposure instrument designed by University of Kentucky Tobacco and Health Research Institute. Three-week-old male guinea pigs were exposed to mainstream, sidestream, or sham smoke, generated twice daily from three cigarettes for 6 weeks. In addition, some animals were kept as room controls for some time. After 6 weeks of smoke exposure, some animals were allowed to recover for 6 weeks without smoke. After 6 weeks of smoking, the plasma retinol levels were lower in both smoke exposed groups when compared to the values in the sham group. Furthermore, in comparison to the sham group, the mainstream and sidestream smoke exposed groups showed a 7.6- and 8.3-fold increase in the levels of lung retinol, respectively. After the 6-week recovery period, plasma retinol of both smoke-exposed groups reached the control levels. In contrast, withdrawal of smoking did not show such an effect on the lung retinol level in both mainstream or sidestream groups. Electronmicroscopy of the lungs showed deleterious alterations in the morphology of the lungs in both mainstream and sidestream groups. Although the mechanism(s) involved in the elevation of retinol content of the lung due to smoke exposure remains to be elucidated, it is of interest that elevation of retinol content and alteration of lung morphology occurred not only in the mainstream smoke exposed but also in the sidestream group. K e y w o r d s - - S m o k i n g , Mainstream, Sidestream, Lung, Vitamin A, Guinea pig, Free radicals
lung. On the other hand, it is well documented that vitamin A (retinol) and its derivatives are involved in the differentiation and maintenance of the proper differentiated state of the lungs. 9 The objective of this research was to determine whether or not mainstream and sidestream cigarette smoke exposure causes modulation in the retinol content of plasma, lung, lung lavage, and liver. As some metabolic characteristics in guinea pigs, such as, for example, those of lung surfactant development, are more similar to humans than in other experimental animals, 1° guinea pigs were used as animal model.
INTRODUCTION
Cigarette smoking has been implicated in the pathogenesis of various types of pulmonary diseases. 1-3 In addition, a recent study indicated that nonsmokers who are exposed to smoke in the household suffer from alterations in lung function. 4 Recent studies also suggest that there is a small but consistent increased risk of lung cancer from passive smoke exposure. 5 We have recently reported that cigarette smoke exposure causes an increase in the free radical-mediated lipid peroxidation potential of erythrocytes in guinea pigs under both mainstream and sidestream conditions. 6 It is possible that the increase in lipid peroxidation of erythrocytes in smoke-exposed animals is associated with the inability of the erythrocytes to quench the free radicals because of deficiency of some antioxidants. In fact, lower levels of carotene and vitamin C have been reported in plasma of smokers. 7'8 Currently, it is not known whether cigarette smoke exposure causes alterations in the vitamin A status in
MATERIALS AND METHODS
Animals
Male guinea pigs (Hartley strain), 3 weeks of age, weighing 2 7 5 - 3 0 0 g were obtained from C a m m Research Laboratories, New Jersey. Animals were divided into four groups: Group RC maintained as room control, Group SH maintained as sham control, Group MS maintained as mainstream smoke-exposed, and Group SS maintained as sidestream smoke-exposed. After an acclimatization period of 7 days, Group RC
Address correspondence to: Salil K. Das, Department of Biochemistry, Meharry Medical College, Nashville, TN 37208, USA.
507
508
S. MUKHERJEE et al.
Table 1. Effects of Mainstream and Sidestream Cigarette Smoke Exposure on Total Particulate Matter (TPM) Inhaled, Blood Carboxyhemoglobin(COHb) Levels, Food Consumption, Body Weight Gain, and Lung Weight of Guinea Pigs Parameters
RC
SH
MS
SS
1.5 (0.2) 313 (17) 34.2 (0.8) 0.77 (0.1)
420 (10) 10.0 (0.3) 189 (16) 33.5 (1.2) 1.29 (0.2)
275 (7) 10.5 (0.2) 158 (18) 33.7 (1.1) 1.36 (0.3)
TPM (mg) COHb (%) Body weight gain (g) Daily food consumption (g) Lung weight (g/100 g body wt.)
317 (9) 35.0 (1.2) 0.79 (0.1)
RC, SH, MS, and SS groups had 3, 8, 8, and 8 animals, respectively. Values are mean and the number in parentheses represents standard error of the mean. Animals were exposed to smoke from 3 cigarettes twice daily for 6 weeks.
contained 3 animals, and other groups contained 13 animals each. The animals were housed in individual stainless steel cages in a temperature-controlled (26°C) 12/12 h light/dark cycle room. They were fed a complete life-cycle diet (Purina Diet #5025) prepared by Purina Mills, Inc., St. Louis, MO. The diet contained 35.9 ppm of carotene, 30 IU/gm of vitamin A acetate, and 50 IU/gm of vitamin E. The animals were allowed to eat and drink ad lib and were weighed at 7-day intervals. Food consumption was recorded daily.
Cigarette smoke exposure conditions Animals of MS and SS groups were exposed twice daily for 6 weeks to either MS or SS smoke from three 1R3F cigarettes in a University of Kentucky Tobacco and Health Research Institute M S - S S smoke exposure system. 6 Briefly, the smoke was generated once each minute by a 2-s 35-ml puff, and drawn into a recycle reservoir for dilution with air before distribution to the exposure chambers. Under these conditions, the animals received smoke for the first 1 6 - 2 0 s followed by air until the next puff was generated 60 s later. Thus, the animals were exposed to fresh cigarette smoke intermittently in a fashion analogous to human smoking. One advantage of this exposure system is that the tendency of animals to withhold breathing during exposures is minimized. The pumpheads of this instrument generate a constant evenpitched sound during exposures instead of the loud clicking sound of the solenoid valve that precedes the generation of puff in some other smoking machines and alerts the animals of smoke influx into the chambers. ~ Additionally, a 7-day break-in period was used in the protocol. During this period, the concentration of smoke in the puff and the number of puffs given during each exposure session were gradually increased. This
acclimatizes the animals and reduces the initial mortality due to smoke. It also discourages the animals from learning to withhold breath during smoke exposures. After acclimatization, the animals were exposed to smoke as described earlier. The cigarettes were smoked to 30.0 mm butt length. Under these conditions, 1R3F cigarettes deliver 18.1 mg of total particulate matter, 1.16 mg of nicotine, and 17.2 mg of CO per cigarette, and average about 9.5 puffs/cigarette. 6 The sham controls received a treatment identical to smoke-exposed groups but in the absence of cigarette smoke to generate similar stress conditions. After 6 weeks of smoke exposure, 5 animals from each of the SH, MS, and SS groups were maintained for an additional 6 weeks without any treatment.
Analysis of indicators of smoke exposure Exposure of animals to cigarette smoke was ascertained by measuring total particulate matter (TPM) inhaled and blood carboxyhemoglobin levels. Estimates of the daily inhaled total and cumulative particulate matter intake were made as described by Griffith and Hancock. 12Briefly, TPM was determined by extracting the filters with 100 ml of acetone, determining the absorbance at 390 nm, and multiplying the results by 100. Control values, determined before smoke exposure, permitted an evaluation of smoking machine performance on a daily basis and for the entire experiment. The difference in control TPM values and the TPM values after exposure gave an estimate of the total TPM dose received by the animals. Carboxyhemoglobin values were obtained on blood samples from the saphenous vein in a hind leg. Twenty microliter blood samples were collected, and the percent of carboxyhemoglobin was determined by the spectrophotometric procedure described by Griffith and Standaferfl 3
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Fig. 1. Transmission electron micrographof lung from a guinea pig exposed to mainstream smoke (3 cigarettes twice daily) for 6 weeks, showing abnormal type II cells protruded into the alveolar space (original magnification × 3400; print magnification X 2.9).
Ultrastructural analysis
Biochemical analysis
Lung samples were fixed with 2.5% glutaraldehyde in 0.1 M Sorensen's phosphate buffer, pH 7.2 for 4 h at 5°C. Tissue samples were washed for 1 h in buffer and treated for 1 h with 1% osmium tetroxide in 0.1 M Sorensen's phosphate buffer. The samples were then dehydrated in 50%, 70%, 95%, and 100% ethanol, exchanged with propylene oxide and allowed to infiltrate overnight in 1:1 propylene oxide-Epon 812 mixture. The tissue was embedded in Epon 812 according to Luft. 14 Thick sections (1 #m) were cut with a glass knife, stained with toluidine blue, and surveyed by light microscopy. For ultrastructural analysis, these sections were then stained with uranyl acetate and lead citrate. A Phillips EM 301 transmission microscope was used for electron microscopic examination.
At the end of the experiment, plasma, lungs, livers, and lung lavages were collected. Lung lavage was performed as described by us earlier. 15 In plasma and lavage samples, retinol was determined by the spectrofluorometric method of Thompson et al. 16Total retinol in the livers and lungs was assessed by the same method after alkaline hydrolysis. ~6 Thus, the values represent a sum of free retinol and retinol present in the form of retinyl esters in these tissues.
Statistical analysis Data were analyzed statistically using the Student's t test. ~7 Data are presented as mean __ standard error. Differences at p < 0.05 were considered significant.
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Fig. 2. Transmission electron micrograph of lung from a guinea pig exposed to sidestream smoke (3 cigarettes twice daily) for 6 weeks, showing abnormal type II cells protruded into the alveolar space (original magnification × 3200; print magnification x 2.8).
RESULTS
Data on total particulate matter inhaled by the animals exposed to MS and SS cigarette smoke for 6 weeks, and the corresponding values on blood carboxyhemoglobin levels, food consumption, body weights, and lung weights are shown in Table 1. Over the 6-week period, each animal from the mainstream and sidestream groups inhaled about 420 _+ 10 and 275 _+ 7 mg total particulate matter, respectively. Cigarette smoke exposure caused a 7-fold increase in the carboxyhemoglobin levels of blood in both smoke-exposed groups in comparison to the sham group. There was no significant difference between mainstream and sidestream groups. When compared to the room control and sham groups, the animals of the mainstream- and sidestream-
smoke exposed groups showed 40% to 50% less body weight gain after 6 weeks. There was no significant difference in body weight gain between the room control and sham groups. In addition, no significant difference in food consumption was noted between the experimental groups. Therefore, the depression in body weight gain in the smoke-exposed groups was not due to loss of appetite. After 6 weeks of smoke exposure, the lung weight of both smoke-exposed groups was significantly higher than that of either room control or sham control group. However, after 6 weeks of recovery, there was no significant difference in this parameter between the different experimental groups. For example, the lung weight was 0.45 _+ 0.08, 0.51 +_ 0.09, 0.57 _+ 0.1, and 0.54 _+ 0.09 g/100 g body weight, in animals of the RC, SH, MS, and SS groups, respectively. Thus the gain of lung weight in the smoke
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Fig. 3. Transmission electron micrograph of lung from a room control guinea pig, showing no protrusion of type II cells into the alveolar space (original magnification x 4,300; print magnification x 2.8). exposed MS or SS group was caused by the smoke and the recovery period was long enough for recovery. Ultrastructural analysis indicated that mainstream smoke exposure caused a massive accumulation of dense, amorphous granulated materials in the alveolar space (see Fig. 1) showing lung injury. Furthermore, irregular expansion of alveolar spaces and release of enlarged type II cells in the alveolar spaces was observed (see Fig. 1). In the sidestream smoke-exposed animals, no accumulation of dense, amorphous granulated material was observed in the alveolar space. On the other hand, abnormally enlarged type 1I cells were protruded into alveolar space similar to those seen in the mainstream smoke-exposed group (Fig. 2). Figure 3 shows the lung morphology of the room control. No protrusion of type II cells was observed in the alveolar space of room control animals. It should be noted that even after 6 weeks of smoke withdrawal, enlarged type II cells were released into the alveolar space of both MS- and SS-exposed groups (see Fig. 4).
Data on the effect of smoke on retinol concentration of plasma, liver, lung, and lung lavage are shown in Table 2. Several alterations were noted. In comparison to the sham group, both the mainstream and sidestream groups showed increase in the levels of retinol in the lungs, 7.6- and 8.3-fold, respectively, after the 6 weeks of smoke exposure. In addition, plasma retinol levels were reduced in both mainstream- and sidestreamsmoke exposed groups. The decrease was higher in the sidestream group (33%) than in the mainstream group (15%). Following 6 weeks of the recovery period, there was no difference in the plasma vitamin A levels between the experimental groups; however, the concentration of vitamin A in lung was still significantly higher in both MS- and SS-groups than that in the sham group. It should further be noted that the sidestream group had about 24% decrease in vitamin A concentration in the lung since the time of withdrawal from smoke exposure. No difference in the vitamin A level in liver and
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Fig. 4. Transmission electron micrograph of lung from a guinea pig exposed to mainstream smoke (3 cigarettes twice daily) for 6 weeks and maintainedfor an additional 6 weeks without any smoke exposure (original magnification× 3200; print magnification X 2.9).
lung between the control, and sham groups was found; however, the plasma vitamin A level was slightly lower in sham group than that of the room control group. The effect in the sham group may be associated with stress, because it is known that stress causes a decrease in the level of retinol in serumJ 8 No significant difference in the vitamin A level in liver, and lung lavage was found between the treatment groups. DISCUSSION Healthy lungs are equipped with structural and nonstructural defense systems to counteract the effects of a variety of potentially injurious agents present in the inspired air. 19 Disease or nutritional deficiency can alter the structural defenses and set up vicious cycles of initial lung injury. The nonstructural, or biochemical,
defense system includes superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and reduced glutathione, which protect the lung from toxic oxygen species and reactive metabolites generated by the pulmonary metabolism of a variety of foreign compounds occurring in the inhaled air. z° Singlet molecular oxygen has been shown to be generated in biological system and is capable of damaging proteins, lipids and DNA. 21 A recent epidemiological study indicates that cigarette smoking is a contributing factor for cancer of the lung. 22 Lung cancer, caused by cigarette smoking, is occurring in all race and sex groups in America. If Americans stopped smoking, 87% of lung cancer deaths could be prevented. 22 Vitamin A plays an essential role in the respiratory tract by influencing differentiation and integrity of the
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Table 2. Effects of Mainstream and Sidestream Cigarette Smoke Exposure on the Vitamin A Levels in Plasma, Liver, Lung, and Lung Lavage of Guinea Pigs Exposure Condition 6Weeks Exposure Samples Plasma (/zg/100 ml) Lung (/~g/g)
RC
SH
MS
SS
SH
MS
SS
67.8 (1.4) 0.5 (0.0)
50.5 (1.1) 0.4 (0.0) 112.5 (4.3) 130.5 (3.2)
42.9 (1.2) 3.1 (0.1) lOl.O (4.6) 124.8 (4.8)
34.0 (2.6) 3.4 (0.2) 108.6 (4.7) 117.0 (4.2)
46.4 (2.8) 0.4 (0.0) 122.9 (5.2) 104.3 (6.7)
50.9 (1.4) 2.6 (0.1) 128.1 (4.8) 123.6 (2.4)
48.2 (1.6) 2.2 (0.1) 117.7 (4.9) 96.4 (8.5)
Lung lavage (#g/mg prot.) Liver (#g/g)
Recovery Period
126.5 (3.6)
RC, SH, MS, and SS groups had 3, 13, 13, and 13 animals, respectively. Out of 13 from each of the SH, MS, and SS groups, 8 animals were sacrificed after 6 weeks of smoke exposure from 3 cigarettes twice daily; remaining 5 animals from each group were maintained for an additional 6 weeks without any treatment. Values are mean, and the number in parenthesis represents standard error of the mean. Following 6 weeks of exposure, the vitamin A levels were significantly higher in lungs and significantly lower in plasma of both MS and SS groups than those of the sham group (p < 0.05). Following 6 weeks of the recovery period, there was no difference in the plasma vitamin A levels between the experimental groups; however, the concentration of vitamin A in lung was still significantly higher in both MS- and SS-groups than that in the sham group (p < 0.0l). Lung and liver values represent total retinol recovered after alkaline hydrolysis of the tissues.
epithelial cells. 9 A d v a n c e d vitamin A deficiency results in replacement o f mucus secreting ciliated epithelium by squamous epithelium in the respiratory tract. 23 Epidemiologic studies have demonstrated an inverse relationship between vitamin A intake and lung cancer rate. a4 Our laboratory previously reported that vitamin A deficiency causes a significant decrease in superoxide dismutase, glutathione peroxidase, and reduced glutathione levels in guinea pig lung.19 Simultaneously, it causes a marked increase in microsomal oxidation. This indicates that vitamin A plays an essential role in defense mechanism o f the cells' removal o f these toxic radicals. 19 W e have also observed that smoking causes a significant modulation o f oxygen defense mechanisms o f guinea pig lung (unpublished observation). This suggests that the lung is capable o f assimilating vitamin A during oxidant exposure providing vitamin A is available in body stores or in the diet. Ultrastructural analysis in our study indicated evidence o f lung injury in guinea pigs exposed to cigarette smoke. Oxidants, such as hydrogen peroxide, present in the cigarette smoke and those produced by the macrophages stimulated by the smoke exposure are believed to play a major role in lung injury, such as chronic bronchitis, emphysema, and inflammatory diseases. 25 Substantial cell proliferation as a response to toxic lung damage is a c o m m o n p h e n o m e n o n in lung pathology. It is now well established that damage and death o f the cells lining most o f the alveolar surface, the type I alveolar cells, m a y be quickly followed by a proliferation o f type II alveolar cells. 26 Retinoids contribute to the maintenance o f the structural integrity
o f respiratory mucosa by controlling proliferation and differentiation o f epithelial cells. 27 Hence, one could expect that there will be change in vitamin A level between normal and toxic lung. In addition, carotenoids and tocopherols can protect tissues against the deleterious effects o f lipid peroxidation by singlet oxygen with their quenching abilities. 21 The chain-breaking reactions o f these antioxidants with lipid peroxyl radicals are generally regarded as being the most important. There is strong evidence that the provitamin A, beta-carotene, is more effective in this.protective effect than vitamin A itself, a4 Our study indicates that the lung o f the guinea pig exposed to both MS and SS cigarette smoke had markedly higher levels o f vitamin A than that of sham and r o o m control groups. One may speculate that the loss of vitamin A from plasma and concomitant increase in its level in the lungs m a y be due to some adaptive mechanism whereby plasma vitamin A is mobilized into the lung. Interestingly enough, it has been reported that smoke exposure causes an increase in the level of vitamin E, another fat soluble vitamin in the lungs. 28 Thus, it is possible that when intake o f vitamin A is adequate, as is the case o f this study, vitamin A m a y minimize or lessen further oxidative damage and/or stimulate cell differentiation which is critical in repairing lung tissue. However, the mechanism how vitamin A is mobilized to the lungs from other parts o f the body remains to be elucidated. Results obtained from this study indicate that sidestream smoke exposure and mainstream smoke exposure have similar effects as far as the recruitment o f
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vitamin A in lung is concerned. We have established earlier that both mainstream and sidestream smoke exposures cause an increase in the production of free radicals in guinea pig erythrocytes.6 Here again we found that the sidestream animals were affected. It appears that the gaseous phase of the smoke, which gives rise in carboxyhemoglobin levels, is mainly responsible for the metabolic reactions rather than the particulate matter, because the animals exposed to the sidestream smoke inhaled 35% less particulate matters than those exposed to the mainstream smoke. Consequently, not only smokers but also nonsmokers exposed passively to cigarette smoke could benefit by adjustment of their vitamin A intake. In conclusion, this study indicates that the vitamin A nutritional status may be a very important determinant of the deleterious effects of cigarette smoke in both smokers and nonsmokers exposed to passive smoke.
9. 10. 11. 12. 13. 14. 15. 16. 17.
Acknowledgements - - This work was supported by grants from
18.
the U.S. Environmental Protection Agency (R-815617), U.S. Public Health Service (HL-14214), and U.S. Agency for International Development (936-5053). The authors are grateful to Mark O. Hunt for excellent technical assistance and Ms. Susmita Chakraborty for proofreading.
19. 20.
REFERENCES
1. Auerbach, O.; Hammond, E. L.; Garfinker, L.; Benante, C. Relation of smoking and age to emphysema. Whole-lung section study. N. Engl. J. Med. 286:853-857; 1972. 2. U.S. Public Health Service (USPHS). Smoking and health: A report of the Surgeon General, DHEW Publication (PHS) 7950066. Washington, DC: U.S. Department of Health, Education, and Welfare, Public Health Service; 1979:10-35. 3. Hunninghake, G. W.; Crystal, R. G. Cigarette smoking and lung destruction. Am. Rev. Respir. Dis. 128:833-838; 1983. 4. Willers, S.; Attewell, R.; Bensryd, I.; Schutz, A.; Skarping, A.; Vahter, M. Exposure to environmental tobacco smoke in the household and urinary cotinine excretion, heavy metals retention and lung function. Arch. Environ. Health. 47:357-363; 1992. 5. Brownson, R. C.; Alavanja, M. C.; Hock, E. T.; Loy, T. S. Passive smoking and lung cancer in nonsmoking women. Am. J. Public Health 82:1525-1530; 1992. 6. Mukherjee, S.; Woods, L.; Weston, Z.; Williams, A. B.; Das, S. K. The effect of mainstream and sidestream cigarette smoke exposure on oxygen defense mechanisms of guinea pig erythrocytes. J. Biochem. Toxicol. 8:119-125; 1993. 7. Pelletier, O. Vitamin C and cigarette smokers. Ann. N. Y. Acad. Sci. 258:156-158; 1975. 8. Chow, C. K.; Thacker, R.; Changchit, C.; Bridges, R. B.; Rehm, S. R.; Humble, J.; Turbek, J. Lower levels of vitamin C and
21.
22. 23.
24.
25.
26. 27. 28.
carotenes in plasma of cigarette smokers. J. Am. Coll. Nutr. 5:305-312; 1986. Chytil, F. The lungs and vitamin A. Am. J. Physiol. 262:(Lung Cell. Mol. Physiol. 6):L517-L527; 1992. Stith, I. E.; Das, S. K. Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes. Biochim. Biophys. Acta 714:250-256; 1982. Gairola, C. G. Free lung cell response of mice and rats to mainstream cigarette smoke exposure. Toxicol. & Appl. Pharmacol. 84:567-575; 1986. Griffith, R. B.; Hancock, R. Simultaneous mainstream-sidestream smoke exposure systems. I. Equipment and procedures. Toxicology 34:123-138; 1985. Griffith, R. B.; Standafer, S. Simultaneous mainstream-sidestream smoke exposure systems. II. The rat exposure system. Toxicology 35:13-24; 1985. Luft, J. H. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414; 1961. Khan, A. Q.; Sikpi, M. O.; Das, S. K. Study of phospholipids of guinea pig lung lavage. Lipids 20:7-10; 1985. Thompson, J. N.; Erdody, P.; Brian, R.; Murray, T. K. l;quorometric determinations of vitamin A in human blood and liver. Biochem. Med. 5:67-89; 1971. Steel, R. G. D.; Torrie, J. H. Principles and procedures of statistics: A biometric approach, 2nd ed. New York: McGraw-Hill Book Company; 1980; 86-121. Takase, S.; Goda, T.; Yokogoshi, H.; Hoshi, T. Changes in vitamin A status following prolonged immobilization (stimulated weightlessness). Life Sci. 51:1459-1466; 1992. Nair, C. R.; Davis, M. M.; Das, S. K. Effect of vitamin A deficiency on pulmonary defense systems of guinea pig lung. Int. J. Vit. Nutr. Res. 58:375-380; 1988. Jenkinson, S. G.; Lawrence, R. D.; Bark, R. F.; Gregory, P. E. Non-selenium-dependent glutathione peroxidase activity in rat lung: Association with lung glutathione S-transferase activity and the effects of hyperoxia. Toxicol. & Appl. Pharmacol. 68:399-404; 1983. Mascio, P. D.; Devassagayam, T. P. A.; Kaiser, S.; Sies, H. Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers. Biochem. Soc. Trans. 18:1054-1056; 1990. Cresanta, J. L. Epidemiology of cancer in the United States. Prim. Care 19:419-441; 1992. Boren, H. G.; Pauley, J.; Wright, E. C.; Kaufmann, D. G.; Smith, J. M.; Harris, C. Cell populations in the hamster tracheal epithelium in relation to vitamin A status. Int. J. Vit. Nutr. Res. 44:382-390; 1974. Goodman, G. E.; Omenn, G. S. Carotene and retinol efficacy trail in heavy cigarette smokers and asbestos-exposed smokers. CARET coinvestigators and staff. Adv. Exp. Med. Biol. 320:137-140; 1992. Baughman, R. P.; Corser, B. C.; Strohofer, S.; Hendricks, D. Spontaneous hydrogen peroxide release from alveolar macrophages of some cigarette smokers. J. Lab. Clin. Med. 107:233237; 1986. Witschi, H.; Cote, M. G. Biochemical pathology of lung damage produced by chemicals. Fed. Proc. 35:89-94; 1976. Jetten, A. M.; Smits, H. Regulation of differentiation of tracheal epithelial cells by retinoids. Ciba FoundSymp 113:61-76; 1985. Airriess, G. R.; Changchit, C.; Chen, L. C.; Chow, C. K. Increased vitamin E levels in the lungs of guinea pigs exposed to mainstream or sidestream smoke. Nutr. Res. 8:653-661; 1988.