Induction of pulmonary metallothionein following oxygen exposure

E N V I R O N M E N T A L RESEARCH 50, 269--278

(1989)

Induction of Pulmonary Metallothionein following Oxygen Exposure BETH A. HART,* GEORGE W. V o s s , * AND JUSTINE S. GARVEYt

*Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, and ?Department of Biology, Syracuse University, Syracuse, New York 13210 Received April 26, 1989 Metallothionein (MT) levels were measured by radioimmunoassay in lungs of animals exposed 0, 3, and 6 days to 85% oxygen. MT levels increased with duration of exposure from 1t2.0 ng/lung in sham air control animals to 872.6 ng/lung in animals exposed for 6 days to oxygen. Gel chromatographic analysis of lung homogenates from oxygen-exposed animals revealed the presence of a copper- and zinc-binding component with an approximate molecular weight of 12,000 Da. It was heat stable and cross-reacted with anti-MT. The induction of pulmonary Cu/Zn-thionein was accompanied by an acute phase response, characterized by elevated serum Cu and ceruloplasmin levels and depressed serum Zn. Total lung Cu and Zn also increased, perhaps as a consequence of normal repairative processes necessitated by the oxidant injury. Increased adrenal weight and coincident thymic atrophy in oxygen-exposed animals suggested the participation of adrenocorticosteroids in the induction process. © 1989 Academic Press, Inc.

INTRODUCTION Metallothionein (MT) is a low-molecular-weight, thiol-rich, metal-binding protein. Although its actual physiological role is not known, MT is thought to function in the detoxification of harmful metals such as cadmium and in the metabolism of the essential metals zinc and copper (Cousins, 1985). MT is an effective free-radical scavenger (Thornalley and Vasak, 1985; Thomas et al., 1986). Cells with high concentrations of MT have increased resistance to radiation (Bakka et al., 1982) suggesting that MT may play a protective role against a variety of stresses, some of which involve free radicals. MT synthesis in the liver is increased by exposure to heavy metals, such as Cd (Webb, 1972), Zn (Bremner and Davies, 1975), and Cu (Winge et al., 1981); infection (Sobocinski et al., 1978); endotoxin injection (DiSilvestro and Cousins, 1984); glucocorticosteroids (Etzel et al., 1979); acute physical stresses of cold, heat, bums, and strenuous exercise (Oh et al., 1978); and chronic stress (Hildalgo et al., 1986, 1988). Considerably less is known about the induction of MT in the lung. Several laboratories have demonstrated increased synthesis of pulmonary MT after repeated inhalation of Cd aereosols (Post et al., 1982, 1984; Sampson et al., 1984; Hart and Garvey, 1986; Hart, 1986). Additional chemical and physical agents capable of MT induction in the lung remain to be established. The lung is constantly exposed to a wide variety of environmental stresses. Perhaps oxidant stresses, such as oxygen or ozone exposure, might mediate the induction of pulmonary MT. We selected the well-established oxygen adaptation 269 0013-9351/89 $3.00 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

270

HART, VOSS, AND GARVEY

model in the rat (Rosenbaum et al., 1969; Crapo et al., 1978) to test this hypothesis. Adult Lewis rats were exposed to 85% oxygen for 0, 3, or 6 days, and the amount of pulmonary MT was quantitated using a sensitive and specific radioimmunoassay (VanderMallie and Garvey, 1978). To further facilitate the physiochemical identification of the MT induced by oxygen stress, we compared the molecular distribution of Cu- and Zn-binding proteins in lung homogenates from oxygen-exposed and air control animals. Results were correlated with (a) serum and lung Cu and Zn concentrations and (b) serum cernloplasmin (CP) oxidase activity. Possible mechanisms to explain the induction of MT by exposure to hyperoxia are discussed. MATERIALS AND METHODS Animals. Male Lewis rats (250-270 g) were purchased from Charles River Supply Co. (Kingston, NY). Animals were maintained at 26-28°C with a 12-hr lightdark cycle and were provided with free access to water and food. Exposure conditions. Exposures were performed in plexiglass-covered cages (40-liter capacity). Animals were maintained, with respect to food, water, lighting, and temperature, as described above. Cages were changed daily. To generate 85% 02, medical grade 100% 02 was mixed with breathing air using Aalborg flow meters (Monsey, NY). An airflow of 2 liters/rain allowed for three air changes per hour. Oxygen concentration was monitored with a Ventronics 02 Analyzer (Temecula, CA). Animal preparations. Animals were anesthetized with pentobarbital (50 mg/kg) and exsanguinated by cardiac puncture. Heparinized plasma was stored at - 73°C for Cu, Zn, and CP oxidase measurements. Lungs were perfused in situ with phosphate-buffered saline (PBS), pH 7.4, containing 0.05% sodium citrate, and stored at -73°C, awaiting MT, metal, and chromatographic analyses. Nonperfused lungs were used for wet and dry weight determinations. Cu and Zn determinations. Minced, lyophilized lungs were digested in 50% nitric acid (Ultrex, J. T. Baker, Phillipsburg, NJ) by reflux heating, in acidcleaned quartz beakers covered with glass coverslips, until less than 1 ml remained. After reconstitution with 3 ml of water, lipids were extracted with 3 ml of chloroform. The aqueous phase was quantitatively transferred to a 10-ml volumetric flask and adjusted to a final nitric acid concentration of 10%. Cu and Zn analysis was performed using a Model 2380 Perkin-Elmer atomic absorption spectrophotometer and atomic absorption standards supplied by Fisher Chemical Co. (Fair Lawn, N J). Matrix interference, determined by the method of standard additions, was less than 5%. Recovery was greater than 97%. Plasma Zn and Cu were determined with an impact bead installed in the burner head of the unit (Makino and Takahara, 1981; Butrimovitz and Purdy, 1977). Gel chromatography. Lungs were finely cut and homogenized in 10 mM Tris buffer (pH 8.6), containing 1 mM dithiothreitol and 0.02% sodium azide (Sigma, St. Louis, MO). A total of 5.0 ml of buffer was added and the tissues were homogenized twice (for 30 sec) at 10,000 rpm using a Biohomogenizer (Biospec Products, Bartletsville, OK). The homogenate was centrifuged for 20 min at 34,000g in a Sorvall superspeed centrifuge. The resultant supernatant fluid was

INDUCTION OF PULMONARY METALLOTHIONEIN

271

centrifuged for 1 hr at 100,000g in a Beckman ultracentrifuge. Temperature was maintained at 4°C. A 0.20-ml portion was analyzed for total metal content. A 3-ml aliquot was applied to a Sephadex G-75 (2.5 x 100 cm) and eluted with 0.01 M Tris buffer (pH 8.6), containing 0.02% sodium azide and 1 mM dithiothreitol. Two hundred fractions, containing 3.65 ml each, were collected and analyzed for Cu, using electrothermal atomization atomic absorption spectroscopy, and Zn, by flame atomic absorption spectroscopy with impact bead. Metal elution profiles were generated by computer and analyzed by planimetry. The Cu and Zn percentage in each peak was multiplied by total cytosolic Cu or Zn content to estimate the amount of metal. Ceruloplasmin. Oxidase activity was measured with the orthodianisidine dihydrochloride method in 0.1 M acetate buffer, pH 5.0. Expressed as international units, results were based on the difference between absorbance readings (at 540 nm) at 5 and 15 rain after addition of substrate (Schosinsky et al., 1974). Metallothionein quantitation. Lung extracts were prepared in 0.01 M Tris buffer, pH 8.6 (Hart and Garvey, 1986) and were quantitated for MT using a radioimmunoassay which employed an antibody (VanderMallie and Garvey, 1978, 1980) prepared in rabbit against liver metallothionein. This antibody cross-reacts with pulmonary MT synthesized in Lewis rats following repeated Cd inhalation (Hart and Garvey, 1986). Statistical analysis. Group differences were assessed by Student's t test. RESULTS

Systemic and pulmonary effects of 85% oxygen. As shown in Table 1, animals exposed to 85% oxygen for 6 days lost an average of 15.1 +- 9.3 g (means -+ SD) of body weight, whereas sham air controls gained 24.8 + 5.9 g. Lung wet and dry weights of oxygen-exposed animals were 1.5 times greater than air controls. Oxygen exposure also affected adrenal and thymus weight, but in an inverse manner; as adrenal weights rose, thymic weights fell. Oxygen-induced alterations in systemic and pulmonary Cu and Zn metabolism. Lung Cu content increased fourfold and Zn content increased twofold after exposure to 85% oxygen for 6 days (Table 2). Increases in lung trace elements were accompanied by changes in plasma Cu and Zn concentrations and in CP activity. Plasma Cu and CP values doubled in response to oxidant stress, whereas plasma Zn was reduced by 23%. The plasma Cu/Zn ratio in oxygen-exposed animals was twice that of controls (Table 2). Quantitation of MT during oxygen exposure. MT was measured in lung homogenates prepared from animals exposed to 85% oxygen for 0, 3, or 6 days (Fig. 1). After 3 days in 85% oxygen, both MT content (ng MT/lung) and concentration (ng MT/g wet wt) doubled compared to sham air control animals. Pulmonary MT continued to rise as a function of exposure duration: after 6 days exposure to 85% oxygen, an eighffold increase occurred. Molecular distribution of pulmonary Cu- and Zn-binding proteins in oxygenand air-exposed animals. Lung cytosols from control and oxygen-exposed animals were chromatographically separated into three Cu- and Zn-binding components (Fig. 2). The components corresponded to molecular weights of 68,000

272

HART, VOSS, AND GARVEY TABLE 1 EFFECT OF 85% OXYGENEXPOSUREON BODYAND ORGANWEIGHTS Parameter Body weight (g) Initial Final Lung wet weight (g) Lung dry weight (g) Lung wet/dry ratio Thymus weight (g) Adrenal weight (g) Thymus/adrenal ratio

Sham air control

Oxygen-exposed~

267.0 (4.9) 291.4 (4.0) i. 1118 (0.0737) 0.2032 (0.0131) 5.44 (0.1559) 0.4353 (0.0177) 0.0351 (0.0032) 12.48 (1.89)

265.8 (8.2)b 250.7c (11.4) 2.0114 (0.1399) 0.3389 (0.0278) 5.91 (0.0980) 0.3440 (0.0568) 0.0415 (0.0034) 8.36 (1.25)

a Animals were exposed for 6 days to 85% oxygen. b The numbers in parentheses are the standard deviations of the means. Oxygen exposed animals have an N = 14 and air controls have an N = 6. c Except for initial body weights, all parameters were significantly different (P < 0.05) for the two treatment groups.

(Peak 1), 34,000 (Peak 2), and 12,000 (Peak 3) Da. Quantitatively m o r e Zn and Cu were associated with the high-molecular-weight c o m p o n e n t than with the other two c o m p o n e n t s in both control and o x y g e n - e x p o s e d animals (Table 3). O x y g e n e x p o s u r e increased the Zn content of all three molecular c o m p o n e n t s but did not change the percentage of Zn associated with each peak. In contrast, oxygen e x p o s u r e altered both the total amount of soluble Cu and the distribution of Cu a m o n g the three molecular components. Lung h o m o g e n a t e s f r o m o x y g e n - e x p o s e d animals had twice as m u c h Cu in the high-molecular-weight c o m p o n e n t and 10 times as m u c h Cu in the tow-molecular-weight c o m p o n e n t c o m p a r e d to sham air controls. Following oxygen exposure, the percentage of total Cu in the lowmolecular-weight c o m p o n e n t increased f r o m 6 to 26%. The c o m p o n e n t s in the low-molecular-weight p e a k (Peak 3) were stable to heating (100°C for 1 min) and showed immunoreactivity to anti-MT.

DISCUSSION The present experimental results suggest that exposure to 85% oxygen induces MT synthesis in the lung. Physiochemical p a r a m e t e r s supporting the identity of this protein include cross-reactivity with polyclonal antibody obtained f r o m rabbits immunized against MT; a molecular weight of approximately 12,000 Da; stability to heating at 100°C for 1 min; and Cu and Zn metal c o m p o n e n t s . T h e increase in pulmonary M T depended upon the duration of oxygen e x p o s u r e in a

273

INDUCTION OF PULMONARY METALLOTHIONEIN TABLE 2 EFFECT OF 85% OXYGEN EXPOSUREON COPPER AND ZINC METABOLISM Parametera Lung Cu (~g Cu/lung) Lung Zn (Izg Zn/lung) Plasma Zn (mg/liter) Plasma Cu (mg/liter) Plasma Cu/Zn Ceruloplasmin oxidase (units/ml of plasma)

Sham air control

Oxygen-exposedb

1.80 (0.01) 17.30 (0.31) 1.26

6.62 (0.98) C 33.10 (1.14) 0.97

(0.15)

(0.12)

1.25

2.31

(0.05)

(0.06)

1.01 (0.14) O.1106 (0.0143)

2.31 (0.22) 0.2442 (0.0359)

a All of the parameters were statistically different (P < 0.05) in the two groups. b Animals were exposed to 85% oxygen for 6 days. c The numbers in parentheses are the standard deviations of the means where N = 6.

manner indicative of a dose-response relationship. After 6 days of oxygen exposure, pulmonary MT levels increased eightfold compared to control levels. Animals exposed to 85% oxygen were under considerable stress as manifested by the loss of 15% of their body weight over the 6 days. Although all the animals survived oxygen exposure, pulmonary damage occurred in the process. The development of pulmonary edema, a final consequence of oxidant injury to the pulmonary capillary endothelial cells, was indicated by increased lung wet weight and increased wet/dry weight ratio in oxygen-exposed animals. The observed increase in lung dry weights was suggestive of extensive restructuring of the lung architecture following oxidant injury (Crapo et al., 1978). Several mechanisms have been suggested to govern the synthesis of pulmonary MT following acute stresses such as cold, heat, burns, and strenuous exercise (Oh et al., 1978). Perhaps one or more of these mechanisms contribute to the induction of pulmonary MT following oxidant stress. One possibility involves stressinduced increases in plasma glucocorticoids, which can induce hepatic MT (Klaassen, 1981). The findings of elevated adrenal weights and thymic atrophy in oxygen-exposed animals were consistent with acute stress and increased levels of adrenocorticoids. Adrenal size is generally considered to be under control of adrenocorticotropic hormone (Masui and Garren, 1970), which stimulates the release of adrenocorticosteroids. Thymic involution, on the other hand, is a wellknown catabolic effect caused by elevations in glucocorticoids (Craddock, 1976). Another possible mechanism for increasing MT synthesis involves glucagon. This hyperglycemic hormone rises with physiological stresses and induces hepatic MT synthesis (Cousins, 1985). Individual inducers of MT synthesis are also known to function synergistically. Glucagon and dexamethasone, a synthetic glucocorticoid, working together augument MT synthesis to a greater extent than either mechanism alone (Kulpers and Cousins, 1984).

274

HART, VOSS, AND GARVEY

c

2O E 12 e~

c

n

0

3

6

0

3

6

800

e~

I-c

Days of oxygen stress

FIG. l. Quantitation of metallothionein in lung homogenates of animals exposed to 85% oxygen for 0, 3, and 6 days.

Stressful stimuli can also trigger an acute phase response. During this pleotropic response to tissue injury and inflammation, the expression of a number of liverspecific genes is induced, probably by the action of interleukin-1 (Epstein, 1984), which increases MT synthesis and lowers serum Zn levels (DiSilvestro and Cousins, 1984). Release of interleukin-1 is thought to cause redistribution of plasma zinc (more in the stored form in the liver) and the release of CP. This Cucontaining protein accounts for at least 90% of the total plasma Cu (Cousins, 1985). Alterations in plasma Cu and CP, itself an acute phase reactant, are closely correlated (Moak and Greenwald, 1984). Oxygen-exposed rats in our study showed a depression of plasma Zn and doubling of Cu and CP levels. Elevation in systemic Cu, caused by the release of CP during hyperoxia, may have led to increased Cu uptake by the lung and induction of MT synthesis (Cousins, 1985; Winge et al., 1981). The physiological role of MT in the lung during exposure to 85% oxygen is speculative. Participation of MTs in normal cellular metabolism and in cellular adaptation to various stresses has been suggested. MT appears to function in the homeostasis of essential metals, such as Cu and Zn. Specifically, MT may serve as a reservoir for Zn and Cu, donating and sequestering these divalent cations to proteins to meet metabolic requirements within the cell (Karin, 1985; Bremner, 1987). The importance of Cu in pulmonary defense is suggested by enhanced toxicity

275

INDUCTION OF PULMONARY METALLOTHIONEIN 90,

90.

80

80

85% 02

AIR

70'

70'

E m

a_ a_

0 u

60

E I]1 e.

,TO 40

MI

60

50 40'

L 30

o

30

u 20

20'

I0

10

0 4O

'

;o

!

;o

0

J

i

!

60

40

ioo

i

80

100

210

2'°I

85% 02

1so

AIR

180

150 120

120 ~-

90

!

2

60 30 0 40

6'o

8'0

'

I00 FRACTION

40

6

I0

i

'|

80

100

NUMBER

FIG. 2. Sephadex G-75 chromatograms of rat lung homogenates showing the molecular distribution of Cu and Zn in oxygen-exposed (85% oxygen for 6 days) and sham air-exposed. Peaks 1, 2, and 3 designate the molecular components that were resolved. Peak 1: fractions 40-57; Peak 2: fractions 58-72; Peak 3: Fractions 73-90.

to hyperoxia exhibited by Cu-deficient rats (Jenkinson et al., 1984). Cu and Zn serve as cofactors for the cytoplasmic form of superoxide dismutase, an antioxidant enzyme believed to limit free-radical-mediated processes initiated by oxygen exposure (Crapo et al., 1980). Reactivation of the cytosolic Zn/Cu superoxide

276

HART, VOSS, AND GARVEY

TABLE 3 EFFECT OF 85% OXYGEN EXPOSURE ON THE MOLECULAR DISTRIBUTION OF SOLUBLE COPPER AND ZINC IN LUNG HOMOGENATES Metal Copper (ng) Total Peak 1 Peak 2 Peak 3 Zinc (ng) Total Peak 1 Peak 2 Peak 3

Sham air control a

Oxygen-exposed

861 681 178 127

(45) b (48) (27) (23)

1802 1164 275 485

(118) (86) (38) (24)

7860 3910 2430 920

(877) (438) (269) (99)

12035 6815 3105 1382

(233) (134) (64) (30)

Animals exposed to 85% oxygen for 6 days. b All parameters were significantly different (P < 0.05) for the two treatment groups. The numbers in parentheses are the standard deviations of the means where N = 3. a

dismutase by Cu-thionein (Geller and Winge, 1980), shown in vitro, suggests another possible role for MT. Lung repairative processes are likely to increase demand for Cu and Zn. The proliferation of type II cells, which contribute to the repair of the alveolar surface damaged by oxygen exposure (Crapo et al., 1980), may be responsible, in part, for the elevation in pulmonary Cu and Zn following oxygen exposure. Zn is required for cell division as a cofactor in enzymes such as thymidylate kinase and DNA polymerase (Duncan and Hurley, 1978). Cu, on the other hand, plays an essential role in tissue repair as a cofactor for lysyl oxidase, an enzyme catalyzing the cross-linking reaction within elastin and collagen (Odel, 1976). With its abundance of thiolate clusters, MT may also contribute, in part, to protecting the lung from damage by oxygen-active species. In vitro experiments have shown that MT reacts rapidly with hydroxyl free radicals formed during oxidative stress and may act as a sacrificial target for oxidative damage. The hydroxyl radical damage to MT apparently occurs at the metal-thiolate clusters, which may be repaired intracellularly by reaction with reduced glutathione (Thornalley and Vasak, 1985). Although MT appears to be a renewable scavenger of hydroxyl free radicals, it is not active toward superoxide radicals, unlike superoxide dismutases or reduced glutathione. The actions of MT, reduced glutathione, and antioxidant enzymes such as superoxide dismutase may function in a cooperative manner. The extent to which MT serves as a scavenger of active oxygen species would depend, in large measure, upon its tissue concentration. Although significant, the 8-fold increase in pulmonary MT produced by exposure to 85% oxygen is much less than the 100-fold increase achieved following exposure to Cd aerosols (Hart, 1986). If MT does protect the lung from hydroxyl radicals generated during oxidant stress, we would predict that Cd-exposed animals, which have greatly elevated lung MT content, would be tolerant to 100% oxygen. We are currently testing this hypothesis.

INDUCTION OF PULMONARY METALLOTHIONEIN

277

ACKNOWLEDGMENTS This investigation was supported by NIH Grants ES 03098 (B.A.H.) and ES 01629 (J.S.G.). We are grateful for the biostatistical analysis of Pamela Vacek and for the editorial assistance of Keith Shute.

REFERENCES Bakka, A., Johnsen, A. S., Endresen, J., and Rugstad, H. E. (1982). Radioresistance in cells with high content of metallothionein. Experientia 38, 381-383. Bremner, I. (1987). Nutritional and physiological significance of metallothionein. Experientia Suppl. 53, 81-107. Bremner, I., and Davies, N. T. (1975). The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem. J. 149, 733-738. Butrimovitz, G. P., and Purdy, W. C. (1977). The determination of zinc in blood plasma by atomic absorption spectrometry. Anal. Chirn. Acta. 94, 63-67. Cousins, R. J. (1985). Absorption, transport, and hepatic metabolism of copper and zinc: Special reference to metaUothionein and ceruloplasmin. Physiol. Rev. 65, 238-308. Craddock, C. G. (1976). Corticosteroid-induced lymphopenia, immunosuppression, and body defense. Ann. Int. Med. 88, 564-566. Crapo, J. D., Barry, B. E., Foscue, H. A., and Shelburne J. (1980). Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Amer. Rev. Respir. Dis. 122, 123-143. Crapo, J. D., Peters-Golden, M., Marsh-Salin, J., and Shelburue, J. S. (1978). Pathologic changes in the lungs of oxygen-adapted rats: a morphometric analysis. Lab. Invest. 39, 640--653. DiSilvestro, R. A., and Cousins, R. J. (1984). Mediation of endotoxin-induced changes in zinc metabolism in rats. Amer. J. Physiol. 247, E436-E441. Duncan, J. R., and Hurley, L. S. (1978). Thymidylate kinase and DNA polymerase activity in normal and zinc deficient developing rat embryos. Proc. Soc. Exp. Biol. Med. 159, 39-43. Epstein, F. H. (1984). Intedeukin-1 and the pathogenesis of the acute phase response. N. Engl. J. Med. 311, 1413-1418. Etzel, K. R., and Cousins, R. J. (1981). Hormonal regulation of liver metallothionein: Zincindependent and synergistic action of glucagon and glucocorticoids. Proc. Soc. Exp. Biol. Med. 167, 233-236. Geller, B. L., and Winge, D. R. (1980). Copper-thionein dependent reconstitution of superoxide dismutase in vitro. Fed. Arner. Soc. Exp. Biol. Monogr. 39, 1473. Hart, B. A. (1986). Cellular and biochemical response of the rat lung to repeated inhalation of cadmium. Toxicol. Appl. Pharmacol. 82, 281-291. Hart, B. A., and Garvey, J. S. (1986). Detection of metallothionein in bronchoalveolar cells and lavage fluid following repeated cadmium inhalation. Environ. Res. 40, 391-398. Hidalgo, J. A., Armario, R. Fos, and Garvey, J. S. (1986). Restraint stress-induced changes in rat liver and serum metallothionein and in zinc metabolism. Experientia 42, 1006-1010. Hidalgo, J. A., Giralt, M., Garvey, J. S., and Armario, A. (1988). Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stressed conditions. Amer. J. Physiol. 254, E71-E78. Jenkinson, S. G., Lawrence, R. A., Grafton, W. D., Gregory, P. E., and McKinney, M. A. (1984). Enhanced pulmonary toxicity in copper-deficient rats exposed to hyperoxia. Fundarn. Appl. Toxicol. 4, 170-177. Karin, M. (1985). Metallothioneins: Proteins in search of function. Cell 41, 9-10. Klaassen, C. D. (1981). Induction of metallothionein by adrenocorticosteroids. Toxicology 20, 275279. Kulpers, P. J., and Cousins, R. J. (1984). Zinc accumulation in rat liver parenchymal cells in primary cultures and response to glucagon and dexamethasone. Fed. Proc. 43, 1403-1410. Ishidato, M., and Metcalf, D. (1963). The pattern of lymphopoiesis in mouse after cortisol administration or adrenalectomy. Aust. J. Exp. Biol. Med. Sci. 41, 637-642. Makino, R., and Takahara, K. (1981). Direction determination of plasma copper and zinc in infants by atomic absorption with discrete nebulization. Clin. Chem. 27, 1445-1451.

278

HART, VOSS, AND GARVEY

Masui, H., and Garren, L. D. (1970). On the mechanism of action of adrenocorticotropic hormone. J. Biol. Chem. 245, 2627-2632. Moak, S. A., and Greenwald, R. A. (1984). Enhancement of rat ceruloplasmin levels by exposure to hyperoxia. Soc. Exp. Biol. Med. 177, 97-103. O'Dell, B. L. (1976). Biochemistry and physiology of copper in vertebrates. In "Trace Elements in Human Health and Disease: Zinc and Copper" (A. S. Prasad, Ed.), Vol. 1, pp. 391--413.Academic Press, New York. Oh, S. H., Deagan, J. T., Whanger, P. D., and Weswig, P. H. (1978). Biological function of metallothionein: Its induction in rats by various stresses. Amer. J. Physiol. 234, E282-E285. Post, C., Johansson, B., and Allenmark, S. (1984). Organ distribution and protein binding of cadmium in autopsy material from heavy smokers. Environ. Res. 34, 29-37. Post, C., Sqibb, K. S., Fowler, B. A., Gardner, D. E., and Hook, G. E. R. (1982). Production of low-molecular weight cadmium-binding proteins in rabbit lung following exposure to cadmium chloride. Biochem. Pharmacol. 31, 2969-2975. Rosenbaum, R. M., Wittner, M., and Lenger, M. (1969). Mitochondrial and other ultrastructural changes in alveolar cells of oxygen-adapted and poisoned rats. Lab. lnvest. 39, 640-653. Rugstad, H. E. (1984). Metallothionein A unique protein with multiple detoxifying properties? Eur. Surg. Res. Suppl. 16, 102-112. Sampson, C. E., Chichester, C. O., Hayes, J. A., and Kagan, H. (1984). Alterations in collagen biosynthesis and in metallothionein in lungs of rats acutely or repeatedly exposed to cadmium chloride aerosol. Amer. Rev. Respir. Dis. 129, 195-205. Schosinsky, K. H., Lemann, H. P., and Beeler, M. F. (1974). Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin. Chem. 20, 1556-1563. Sobocinski, P. Z., Canterbury, W. J., Mapes, C. A., and Dinterman, R. E. (1978). Involvement of hepatic metallothionein in hypozincemia associated with bacterial infection. Amer. J. Physiol. 234, E399-406. Thomas, J. P., Backowski, G. J., and Ghirotti, A. W. (1986). Inhibition of cell membrane lipid peroxidation by Cd and Zn metallothioneins. Biochim. Biophys. Acta 884, 448-461. Thornalley, P. J., and Vasak, M. (1985). Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36-44. VanderMallie, R. J., and Garvey, J. S. (1978). Production and study of an antibody produced against rat cadmium thionein. Immunochemistry 15, 357-368. VanderMallie, R. J., and Garvey, J. S. (1980). Radioimmunoassay of metallothioneins. J. Biol. Chem. 254, 8416--8421. Webb, M. (1972). Binding of cadmium ions by rat liver and kidney. Biochem. Pharmacol. 21, 27512765. Winge, D. R. R., Geller, B. L., and Garvey, J. S. (1981). Isolation of copper-thionein from rat liver. Arch. Biochem. Biophys. 208, 160-166.