ENVIRONMENTAL RESEARCH 41, 302-308 (1986)

Neonatal Developmental Pattern of Superoxide Dismutase and Aniline Hydroxylase in Rat Lung

POONAM KAKKAR, FARHAT N. JAFFERY, AND P. N. VISWANATHAN

Industrial Toxicology Research Centre, P.O. Box 80, M. G. Marg, Lucknow-226001, Uttar Pradesh, India

Received September 10, 1984

The developmental biology of superoxide dismutase and aniline hydroxylase was fol- lowed in rat lungs from prenatal stage to 3 months old. Total superoxide dismutase activity as determined by spectrophotometry as well as electrophoresis was high in the prenatal rat lung, decreased in the first 24 hr postpartum, increased within 7 days, and then decreased gradually to adult levels. On polyacrylamide gel electrophoresis only two isozymic forms of superoxide dismutase were located as achromatic zones in the fetal lung. In the adult rat lung, there were three molecular forms of superoxide dismutase, two in the postmitochon- drial supernatant and one in the mitochondrial fraction. Unlike superoxide dismutase, ani- line hydroxylase was detectable only after 5 days of age and the activity exhibited a gradual increase afterward up to 1 month of age. The developmental pattern of superoxide dismu- tase and aniline hydroxylase activities in lung may be significant in understanding the mech- anism of body defenses and their regulatory modulations in response to toxic air pollutants and environmental stress. © 1986 Academic Press, Inc.

INTRODUCTION

The understanding of regulation of metabolism in the lung, in relation to physi- ological and stress conditions, is an important aspect of environmental health research, such as toxicity of airborne xenobiotics and high altitude physiology (Lensen, 1969). Therefore, study of the developmental pattern of key biochemical parameters involved in structure and function is of paramount importance. In spite of considerable information on anatomical and histological (Hodson, 1977) aspects of lung development comparatively very little is known about the alter- ations in the biochemical machinery during neonatal development, excepting sur- factant turnover (Brumley et al., 1967). In the transition from the fetal stage with maternal blood as the sole source of oxygen to the postnatal stage with direct supply of oxygen, the lung undergoes a sudden change. This prompted a study of the developmental pattern of superoxide dismutase (SOD, EC 1.15.1.1) in the lung. Aniline hydroxylase (EC 1.14.1.1) was also assayed during neonatal devel- opment in order to follow the development of biotransformation mechanisms in the lung.

MATERIALS AND METHODS

Chemicals . Phenazine methosulfate, (PMS), NADH, nitroblue tetrazolium (NBT), and other biochemicals were procured from Sigma Chemical Company. Other chemicals used were either BDH AnalR or equivalents.

Animals. Pregnant albino rats of ITRC inbred colony maintained under stan-

0013-9351/86 $3.00 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

302

NEONATAL PATTERN OF SOME ENZYMES IN RAT LUNGS 303

dard conditions were separated. Litters were collected based on their age, taking time of birth as 0 hr. For some experiments, the animals were dissected at the fetal stage.

Enzyme preparations. Rats were killed by cutting the jugular vein to avoid blood clotting in lungs. Lungs were then cleared of adhereni tissue, weighed, minced, and homogenized in 0.25 M sucrose using a Potter-Elvehjem motor- driven homogenizer at 0-4°C. The homogenized material was filtered through a twofold muslin cloth to remove connective tissues and diluted with 0.25 M su- crose to 30% w/v. This suspension was centrifuged at 700g for 10 min to remove cells and nuclear debris. The supernatant was then centrifuged at 12,000g for 30 min to separate mitochondria (all centrifugations were carried out at 0-4°C). The pellet was suspended in a minimum amount of distilled water and kept for freezing and thawing (Morton, 1955). The postmitochondrial fraction was assayed for aniline hydroxylase activity.

The cytosolic fraction was diluted to 10% w/v. The mitochondrial fraction was sonicated at 25 kcs for 5 min and centrifuged to solubilize superoxide dismutase. Both fractions were separately salted out by 90% saturation of ammonium sulfate (Green and Hughes, 1955). This was done to remove any inhibitory substances present in the crude preparation that may hinder the SOD activity assay. Both fractions were then centrifuged, dialyzed against distilled water for 12 hr, and centrifuged again. The supernatants thus obtained were then tested for SOD ac- tivity. All operations were carried out under c01d conditions (0-4°C).

Measurement ofsuperoxide dismutase activity. SOD activity was measured in mitochondrial as well as cytosolic preparations by the spectrophotometric and electrophoretic methods of Nishikimi et al. (1972). The assay mixture contained 1.2 ml sodium pyrophosphate buffer, pH 8.3 (0.052 M), 0.1 ml phenazine metho- sulfate (186 txM), 0.3 ml nitroblue tetrazolium (300 IXM), 0.2 ml NADH (780 IXM), enzyme preparation (0.15-0.9 mg/ml), and water in a total volume of 3 ml. Op- tical density was measured on a Unicam SP 500 spectrophotometer at 560 nm, at room temperature. One unit of the enzymatic activity is expressed as the enzyme concentration required to inhibit the N B T - P M S - N A D H reaction system by 50% (Nishikimi et al., 1972).

Polyacrylamide gel electrophoresis. Ammonium sulfate fractionated and dia- lyzed samples of mitochondrial (0.025 mg protein) as well as cytosolic fractions (0.05 mg protein) were separately subjected to polyacrylamide gel electrophoresis using a Tris-glycine tank buffer, pH 8.3. Fractions were applied on 7.5% poly- acrylamide gels and SOD activity was located on gels as achromatic zones by the inhibition of the N A D H - N B T - P M S reaction system (Nishikimi et al., 1972). All operations were carried out under cold conditions (0-4°C).

Measurement o f aniline hydroxylase activity. The method of Imai et al. (1966) based on the spectrophotometric estimation of p-aminophenol formed was used. The enzyme was incubated at 37°C for 30 min with NADP (3.2 mM), glucose 6-phosphate (30 raM), nicotinamide (80 raM), and magnesium chloride (25 mN) in 0.2 M phosphate buffer, pH 7.4, making the total volume to 1 ml. Protein was precipitated with 20% trichloroacetic acid (TCA) and centrifuged. To the superna- tant, 10% sodium carbonate was added followed by 2% phenolic sodium hy-

304 KAKKAR, JAFFERY, AND VISWANATHAN

TABLE 1 PULMONARY ANILINE HYDROXYLASE ACTIVITY AT DIFFERENT STAGES OF DEVELOPMENT

Age at sacrifice p-Aminophenol formed (days) (nmole/min/mg protein)

Prenatal N.D. 1 0.048 ± 0.003 3 0.051 _+ 0.004 5 0.130 _+ 0.020

12 0.242 _+ 0.025 20 0.504 _+ 0.021 30 0.570 ± 0.032 60 0.563 _+ 0.021 90 0.559 _4- 0.063

Note. Values are averages _+ SD of 5 determinations in each case. N.D., not detectable.

droxide, and the aminophenol color formed was measured at 630 nm on a Unicam SP 500 spectrophotometer.

Protein. Protein was measured by the method of Lowry et al. (1951) with bo- vine serum albumin as standard.

RESULTS

Aniline hydroxylase activity (Table 1) was absent in prenatal lungs and was found only in trace amounts in l- to 3-day-old rats. Up to 30 days postpartum, aniline hydroxylase activity increased progressively, after which it remained con- stant to the adult level. There was a considerable increase in the recovery of protein in the mitochondrial fraction of lungs from animals 1 day postpartum compared to the fetal lung, indicating changes in the mitochondrial status (Table 2). Mitrochondrial Mn-containing SOD activity was high in lungs from fetal an- imals but suddenly decreased. From Day 1 to Day 7 it again increased, beyond

TABLE 2 MITOCHONDRIAL SUPEROXIDE DISMUTASE ACTIVITY IN LUNGS OF RATS

DURING EARLY DEVELOPMENT

Age at sacrifice Protein Specific activity (days) (mg/ml) (units/min/mg protein)

Prenatal 1.07 _+ 0.022 32.29 ± 0.847 1 1.65 ± 0.034 15.38 - 0.240 3 1.57 ± 0.027 24.28 ± 0.487 5 1.30 ± 0.025 27.33 _+ 0.482 7 1.25 + 0.018 37.98 -+ 0.383

12 1.60 ± 0.030 23.52 -+ 0.254 20 1.54 _+ 0.019 15.83 _+ 0.753 30 1.77 ± 0.028 11.20 _+ 0.422 60 1.90 _+ 0.033 14.33 ± 0.399 90 1.75 _+ 0.020 13.79 -+ 0.668

Note. Values are averages ± SD of 5 determinations in each case.

N E O N A T A L PATTERN OF SOME ENZYMES IN RAT LUNGS 305

TABLE 3 CYTOSOLIC SUPEROXIDE DISMUTASE IN LUNGS OF RATS DURING EARLY DEVELOPMENTAL STAGES

Age at sacrifice Protein Specific activity (days) (mg/ml) (units/rain/rag protein)

Prenatal 4.87 +_ 0.121 32.64 +- 0.859 1 10.27 +- 0.160 9.01 _+ 0.489 3 12.02 _+ 0.106 8.98 -+ 0.229 5 11.10 _+ 0.141 10.47 _+ 0.338 7 8.73 _+ 0.115 33.74 -- 0.120

12 9.67 +-- 0.106 25.75 _+ 0.548 20 10.27 _+ 0.102 10.57 _+ 0.166 30 11.25 _+ 0.112 10.12 _+ 0.229 60 13.50 _+ 0.125 9.76 +- 0.282 90 13.20 +_ 0.132 13.13 +-- 0.502

Note. Values are averages +_ SD of 5 determinations in each case.

which the activity gradually decreased to adult levels which are very nearly the same as observed at 1 day postpartum (Table 2). Cytosolic Cu-Zn SOD also had high activity at prenatal stage, but decreased immediately and then increased up to 7 days (Table 3). Therefore, a biphasic curve with high fetal levels of SOD and high levels of SOD from 3 to 12 days postpartum is suggested for both mitochon- drial and cytosolic SOD. Nonmitochondrial protein (postmitochondrial superna- tant) also increased during early development, possibly indicating increased con- tent of endoplasmic reticulum. Polyacrylamide gel electrophoresis for SOD iso- zymes showed that the mitochondrial isozyme and one cytosolic isozyme were found in up to 20-day-old lungs (Figs. 1 and 2). An additional cytosolic isozyme started appearing in the lungs of 1-month-old rats and was present in 2- and 3- month-old rats (Figs. 2 and 3). The difference in electrophoretic mobility between the mitochondrial and cytosolic isozymes of SOD indicates their different phys- ical natures. Since the purpose of the study was to follow the developmental

. . . . . . .

m m

I - - A "B C D E

FIG. 1. Polyacrylamide gel electrophoresis of partially purified mitochondrial preparations (0.025 mg) of (A) prenatal, (B) 3-day, (C) 7-day, (D) 20-day, and (E) 30-day-old rat lung. Arrows indicate (I) site of application and (II) dye front. For details see text.

306 KAKKAR, JAFFERY, AND VISWANATHAN

A B C D E F

m e

• m m • • m

i

i Fro. 2. Polyacrylamide gel electrophoresis of partially purified postmitochondrial preparations

(0.050 mg) of (A) prenatal, (B) 1-day (C) 3-day, (D) 7-day, (E) 20-day, and (F) 30-day-old rat lung. Arrows indicate (I) site of application and (II) dye front. For details see text.

pattern of SOD, the isozymic pattern in animals beyond reaching adulthood was not studied,

DISCUSSION

Developmental patterns of both superoxide dismutase and aniline hydroxylase Show a Significant increase in the initial postnatal (Days 1-5) stages. In the case

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A B

FIG. 3. Polyacrylamide gel electrophoresis of (A) partially purified postmitochondrial preparation (0.040 rag) and (B) a mixture of partially purified mitochondrial (0.025 rag) and cytosolic (0.025 rag) preparations of adult rat lung. Arrows indicate (I) site of application and (lI) dye front. For details see text.

NEONATAL PATTERN OF SOME ENZYMES IN RAT LUNGS 307

of aniline hydroxylase, significant amounts of enzyme activity in rat lungs were not observed until 5 days after birth, in agreement with the developmental pattern of cytochrome P-450 content in lungs (Philpot et al., 1977). Thus even at early developmental stages, pulmonary tissue establishes defense mechanisms for bio- transformation of xenobiotics. This may be significant in relation to the alter- ations in toxic response to environmental air pollutants in lung. Tyson et al. (1982) have reported such variations in oxidant toxicity, which are correlated to changes in glutathione levels. The differentiation processes, functional organiza- tion of endoplasmic reticulum and its microsomal catalysts, along with direct ex- posure to air and thereby formation of hydroxyl radical, reach optimum by 5 days, so that aniline hydroxylase can be detected only after that.

The high SOD activity present in prenatal lung markedly declined in the first 24 hr after birth. There is a possibility that fetal lung SOD is supplied by the mother's blood. The abundant oxygenated maternal blood supply may lead to higher levels of superoxide radicals in the collapsed fetal lung, which is free from gases. Also bilirubin and oxyhemoglobin (Misra and Fridovich, 1972) are known to activate free radical formation. A high level of SOD could counteract the ef- fects of these free radicals. After birth, this stress by free radicals may be re- duced, leading to lowered SOD in 24 hr. Up to 7 days postpartum, the increase in lung SOD activity is relatively fast. During initial development and differentia- tion, metabolic activity is triggered. This can lead to a large number of free rad- icals being produced and in its defense, SOD is induced. Once the development of respiratory physiology is complete, both mitochondrial and cytosolic SOD levels reach adult values.

Appearance of an additional isozymic form of cytosolic SOD after 30 days is interesting. This additional form of SOD may be due to a metabolic reaction taking place in the adult rat lung but absent in the initial stages of the developing lung which induces a new isozyme to protect against additional production of superoxide radical. Thus the metabolic regulation of SOD isozymes in the devel- oping lung could prove an exciting field of study, especially in defense against free-radical-forming air pollutants. Since biotransformation mechanisms also follow a similar pattern, any relation between SOD and mixed function oxidases could be of interest in pulmonary defense against airborne toxicants.

ACKNOWLEDGMENTS

Thanks are due to Dr. G. B. Singh for his interest in the work. This investigation was financed in part by the Department of Environment, New Delhi.

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