JOURNAL OF APPLIED TOXICOLOGY, VOL. 7(1), 7-13 (1987)

Induction of Superoxide Dismutase Isozymes in Rabbit Lung due to Aniline Exposure

Poonam Kakkar? and P. N. Viswanathan Ecotoxicology Project, Industrial Toxicology Research Centre, Post Box 80, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India

Key words: superoxide dismutase; isozyme; induction; toxicity; aniline; lung oxidants; inhalation; rabbit; free radicals.

Single exposure of rabbits to aniline vapors caused induction of superoxide dismutase isozymes and activity as evidenced by the incorporation of radioactive amino acids and prosthetic group metals into enzymatic protein zones of electrophoreograms. Induced of a new cytosolic isozyme following aniline exposure was also evident. Xenobiotic stress indicated a close relation between free radical processes and lung biotransformation mechanisms.

~

INTRODUCTION Treatment with radioactive isotopes and aniline ~

Free radical mediated processes utilizing active oxygen species are involved in the pulmonary response to stress arising from several environmental factors including airborne toxicants.’,’

Active oxygen species are also involved in lipid per~xidation,~-~ membrane damage and biotransfor- mation proce~ses.~,’ In spite of the importance of 0; and the defence against it through superoxide dismu- tase (SOD, EC 1.15.1.1) in pulmonary toxicology, the functional status of SOD isozymes and their regulatory mechanisms in healthy and diseased lung are not fully characterized biochemically. A parallel response of aniline hydroxylase and of SOD isozymes in lung dur- ing fetal and neonatal stages8 indicated a possible func- tional relationship between them. Aniline, which is an occupational toxicant in the chemical industry (causing pulmonary oedema and methaemoglobinemia9Jo) undergoes hydroxylation in the lung,’ suggesting a possible involvement of active oxygen radicals. In view of the above, the functional status of SOD isozymes in rabbit lung and their induction due to exposure to aniline vapors was studied.

Three groups of four rabbits each were intravenously administerd 40 pCi 54Mn, 40 pCi 65Zn and 20 pCi 14C respectively. Radioactive Mn and Zn were used because they are the prosthetic groups of SOD isozymes. Radioactive amino acid incorporation is a routine technique in studying enzyme induction.

Another three groups of four rabbits each were exposed to 10 ml aniline as vapor form immediately after administration of the isotopes. The exposure to aniline vapor was in a 175 1 glass chamber for 30 min, at room temperature. The aniline vapor level in the chamber was not monitored. The dose level and dura- tion of exposure were selected on the basis of pilot experiments, to ensure non-symptomatic exposure. The purpose of the study was to determine whether short-term exposure to a subtoxic dose of aniline resulted in any change in the superoxide dismutase level in the lung. Controls comprise untreated rabbits, and rabbits exposed to aniline vapor only.

Preparation of subcellular fractions of lung

EXPERIMENTAL

Chemicals

All biochemicals used were procured from Sigmp Chemical Co., USA. Other chemicals were either BDH AnalR or E. Merck extra pure. Isotopes (i.e. 54Mn Cl,, 65Zn C1, and 14C, Chlorella protein hydrolysate) were obtained from the Isotopic Division, Bhabha Atomic Research Centre, Bombay.

Animals

Male albino rabbits maintained under standard condi- tions and weighing 2-2.5 kg were obtained from the Industrial Toxicology Research Centre Animal Colony. ?Author to whom correspondence should be addressed.

02~37X/87/010007-06$05 .OO 0 1987 by John Wiley & Sons, Ltd

The rabbits were killed 24 h after aniline exposure, by injecting air intravenously, and their lungs were removed immediately. The lungs were washed thoroughly with 0.25 M sucrose solution, cleared of adhering materials, blotted on paper and weighed. Lung homogenates [in 0.25 M sucrose to 30% (w/v)] were prepared using a PotterElvehjem homogenizer. Mitochrondrial and post-mitochondria1 fractions were separated by centrifugation of the post-nuclear super- natant at 10,000 X g for 20 min. The mitochondria1 pellet was washed on the centrifuge with fresh medium and suspended in 0.25 M sucrose solution. All the operations were carried out at 2-6°C.

Measurement of radioactivity in subcellular fractions

Small pieces of lung tissue were weighed and the radioactivity was measured on an LKB Ultra Gamma I1 1280 counter. After weighing and homogenization of

Received 4 November 1985 Accepted 3 April 1986

8 P. KAKKAR A N D P. N. VISWANATHAN

the remaining tissue, homogenate, nuclear debris, mito- chondrial and post-mitochondrial fractions were counted for radioactivity on an LKB Ultra Gamma I1 1280, in the cases of "Mn and 65Zn. In the case of 14C treated animals, radioactivity in subcellular fractions was measured on an LKB rack beta counter (Model 1215/1216). The composition of the scintillation cocktail was 52 g napthalene, 2.25 g 2,5-diphenyloxa- zole, 65 mg 1,4-bis 2-( 5-phenyloxazolyl) benzene, 250 ml dioxane, 150 ml methanol and 250 ml toluene. A standard of 14C protein hydrolysate was similarly treated.

untreated). Activity of SOD on the gels was located as achromatic zones, according to the method of Nishi- kimi etal.I3

Measurement of radioactivity in the achromatic zones of polyacrylamide gels

Radioactivity was measured by cutting the gels into 0.5 cm pieces of equal size and, in the case of 54h4n and 65Zn treated animals, performing y counts. For 14C counting, gel pieces were dissolved in H,O, at 70°C for 12 h and scintillation fluid was added. The resultant solution was counted.

Partial purification of SOD Enzyme assay

Since activity could not be detected in the crude homogenate, mitochondrial or cytosol fractions, due to interfering factors, the SOD enzyme was partially puri- fied by ammonium sulfate fractionation.'' The post- mitochondrial supernatant of the lung was saturated with 90% ammonium sulfate, the mixture being contin- uously, gently stirred. The mitochondrial fraction, maintained at ice cold temperatures, was sonicated intermittently at 25 KCs for 5 min and centrifuged before ammonium sulfate fractionation (90% satura- tion). Both samples were separately centrifuged at 10 000 x g for 20 min and the sediments, suspended in water, were dialyzed against distilled water for 12 h. Dialyzed samples were centrifuged and the superna- tants were assayed for SOD activity.

Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured by the inhibition of the nicotinamide adenine dinucleotide (reduced)-phenazine methosulfate-nitro- blue tetrazolium reaction system as described by Nishi- kimi et a l l 3 The assay system consisted of 1.2 ml 0.052 M sodium pyrophosphate buffer at pH 8.3, 0.1 ml 186 p~ solution of phenazine methosulfate, 0.3 ml300 ,UM solution of nitroblue tetrazolium, 0.2 ml 780 p~ solution of NADH and 1.2 ml distilled water to make up the volume to 3 ml so that O.D. of 0.14k0.02 per minute was produced. In this system, SOD was added to measure the enzyme activity. Opti- cal density was measured on a Unicam SP 500 spectro- photometer, at room temperature. One unit of the enzyme is equivalent to 50% inhibition in the formazan formation in 1 min at room temperature.

Polyacrylamide gel electrophoresis Protein

Polyacrylamide gel electrophoresis was performed on the partially purified samples of mitochondrial and post mitochondrial SOD of all animals (treated and

Protein in different subcellular fractions was deter- mined by the method of Lowry et al.,14 after precipitat-

Table 1. Distribution of radioactivity (counts per minutehg protein) in the different subcellular fractions

54Mn 652n '4C

Fraction Control Experimental Control Experimental Control Experimental

Homogenate

Nuclear debris

Post-mitochondria1 fraction

Mitochondria1 fraction

Partially purified post-mitochondria1 fraction

Partially purified mitochondrial

98f8 105f10

125f21 149+18

129k11 214 + 27 171 t13 210f17

144f12 98f7

( P <0.3)

( P <O.OOl)

( P <O.Ol)

( P <O.Ol)

( P <0.001)

214k14 290f11 ( P < 0.001)

77+7 90f4

79+5 94f8

58k7 80f5

181 f28 205f11

81 f10 154f18

( P <O.Ol)

( P CO.01)

( P <0.001)

( P CO.1)

( P < 0.001)

276f15 200f19 ( P <O.OOl)

58+5 81 f5

65+7 80f4

90k10 97f8 ( P < 0.3)

99f6 267 f 22

63f10 97f12

( P < 0.001)

( P < 0.001)

( P < 0.001)

(P < 0.001)

62f12 121 f15 ( P < 0.001 1

fraction

(cpm/g) ( P <O.OOl) ( P < 0.001) Whole tissues 3348 + 248 2114k105 6749 f 74 4982 f 97 - -

All the values are arithmetic mean f SD of four separate determinations in each group. P value as compared with the corresponding control is given within parentheses.

9 SOD ISOZY MES AFTER ANILINE EXPOSURE

ing with 10% TCA. Bovine serum albumin was used as the standard. Data are expressed as mg/g fresh weight of tissue.

Statistical analysis

In each group, estimations were done on four separate animals and the statistical significance evaluated by the students’ ‘t’ test. In the electrophoreograms radioactive counts for one representative of the group is given. The same pattern was generally observed in the other three animals.

RESULTS ~

Aniline-exposed lung showed distinctly lower counts of 54Mn and 65Zn as compared with controls in terms oi total tissue mass (Table 1). This may be because of altered transport of the metals from the lung. However, there was an increase in the counts of all the subfrac- tions of %ln- and aniline-treated animals, except for the partial purified post-mitochondria1 fraction (active fraction) which showed a decreased count. In the case of 65Zn- and aniline-treated animals a similar pattern was obtained except that the partially purified mito- chondria] fraction showed a slightly decreased count whilst in the partially purified post-mitochondrial frac- tion it was increased. The enrichment of 54Mn in mito- chondrial protein and of 65Zn in the post-mitochon- drial protein, during salting out and dialysis, is indicative of the induction of the respective metal-

containing proteins. In 14C + aniline treated rabbits, a regular increase of counts in all the fractions was observed, indicating synthesis of additional protein. In spite of this, the protein content showed a decrease, expressed in terms of fresh weight, which may be attri- buted to retention of water caused by oedema or inflammation.

A two-fold increase in both mitochondrial and cyto- solic SOD was observed following aniline exposure as compared with the corresponding fractions from unex- posed controls (Table 2). When s4Mn was given, a simi- lar pattern was observed. With 65Zn, the induction was even more marked, leading to 3.7 and 5.0 fold increases for mitochondrial and cytosolic components respectively. Induction of SOD was also distinct in the I4C treated aniline exposed animals, leading to 2.5 and 3.1 fold enhancement. The increase in specific activity was of a higher magnitude than was accountable for by a decrease in protein content caused by the aniline- induced oedema. Thus induction of SOD following aniline exposure is a true phenomenon and not due to variations in protein content.

To test induction of specific components, the enzyme samples were subjected to polyacrylamide gel electrophoresis and stained for SOD. Figure 1 shows the isozyme polymorphism in the various cases. The major change was a new (third isozyme) form in 65Zn- treated animals induced by aniline exposure. Evidence for a newly synthesised enzyme could be obtained in both mitochondrial and soluble components by measurement of the radioactivity of the achromatic zones (Table 3). Low counts were detectable in the enzyme zones because of the small amounts applied. In

Table 2. SOD activity of mitochondrial and post-mitochondria1 fractions of rab- bit lung

Fractions

Untreated C M E M CPMF EPMF

C M E M CPMF EPMF

C M E M CPMF EPMF

C M E M CPMF EPMF

54Mn exposed

65Zn exposed

14C aminoacid exposed

Protein Specific activity of SOD (mg/g fresh weight) (units/mg protein)

2.1 7 + 0.1 3 1.46 k0.12 ( P < 0.02) 13.98 k 0.37 11.58f0.42 ( P <0.001)

2.59 f 0.09 1.43 f 0.08 ( P < 0.001) 16.41 k 0.63 13.53f0.57 ( P <O.OOl)

2.42 f 0.1 0

16.53 & 0.52 13.29f0.34 (P<O.OOl)

2.00 f 0.09 ( P < 0.001)

3.26f0.13 1.76f0.11 ( P <0.001) 17.01 f 0.48 13.90+0.36 ( P < 0.01)

20.1 f 0.37 39.3f0.52 (P<O.OOl) 17.8 f 0.58 34.9f0.95 ( P <O.OOl)

23.9 f 0.76 50.5 f 1.75 ( P < 0.001) 16.5 k 0.63 36.9 f 0.25 ( P < 0.001 )

25.3 k 0.66 93.3k0.79 ( P <O.OOl) 10.1 f 0.51 50.0f0.12 (P<O.OOl)

13.6 f 0.38 34.0f1.02 ( P <0.001) 23.8 f 0.18 73.8k0.69 (P<O.OOl)

All the values are arithmetic rneanfSD of four separate determinations in each group. PMF: post-mitochondrial; C, control; E, aniline exposed; M, mitochondrial. p value as compared with the corresponding control is given within parentheses.

P. KAKKAR A N D P. N. VISWANATHAN

(B) Polyacrylamide disc gel electrophoresis of the partially puri- fied mitochondrial preparations of (a) 65Zn treated and (b) 65Zn +aniline treated rabbit lung. 0 and D indicate origin of sam- ple and dye front, respectively. The achromatic zones correspond to the SOD isozymes.

Figure 1(A). POlYacrYlamide disc gel electrophoresis of the Par- tially purified (a) post-mitochondria1 and (b) mitochondrial prepar- ations of 54Mn treated rabbit lung. The number and position of isozymes remained the same in the case of aniline exposed (%Mn treated) animals as well as '"C treated aniline exposed and unex- posed rabbit lungs. 0 and D indicate origin of sample and dye front, respectively. The achromatic zones correspond to SOD isozymes.

the case of 54Mn the counts in both mitochondrial and cytosolic achromatic zones were slightly higher as shown in Figure 2(a) and 2(b) respectively. With 65Zn there was no increase in mitochrondrial components, but cytosolic counts increased in all bands. A new band was also detected in both cases [Figures l(b), l(c), 3(a) and 3(b)]. Some radioactive carbon could also be detected in the same regions of mitochondrial and cytosolic SOD bands as shown in Figure 4. Counts in the other parts of gels may be due to the adsorption of the metal on the gel itself or interchange of metal between the proteins. Also, since the preparations were only partially purified, other proteins containing these metals may be showing their peaks in the graph of gel counts. As such it is difficult to determine the specific metal content of individual bands. A preference for Mn in mitochondrial SOD and Zn in cytosolic SOD is indi- cated.

(C) Polyacrylamide disc gel electrophoresis of the partially puri- DISCUSSION

~~

fied post-mitochondria1 preparations of (a) "Zn treated and (b) 65Zn +aniline treated rabbit lung. 0 and D. indicate origin of sam- ple and dye front, respectively. The achromatic zones correspond

Enhanced activity Of and the detection Of both carbon and prosthetic group isotopic labels in the enzyme zones of electrophoreograms clearly demon- to SOD isoqmes.

SOD ISOZYMES AFTER ANILINE EXPOSURE

150- c 3 z

130- 0 . ," 110- z 3 0

90-

70

11

-

Table 3. Radioactivity in SOD zones of electrophoreograrns

250-

230-

210

190-

Mitochondria1 SOD Post-mitochondria1 SOD CPM CPM

Treatment Zone1 Zone2 Zone3 Zone1 Zone2 Zone3

-

54Mn 12 15 - 12 12 - 54Mn+Aniline 15 22 - 29 31 - h5Zn 46 38 - 24 32 - 65Zn +Aniline 31 51 32 53 40 64 14C 126 108 - 146 162 - 14C+Anitine 202 162 - 200 176 -

170- c 3 z

150- .-l . 2 130- I 3 0 " Ira-

90

70

so

CPM, Counts per minute. The gels were cut into sections and counted as described in text. The data represent the value for the achromatic zones.

- - -

202 0 0 2 1 6 8 10 12 1.4 16

FRACTION N U M B E R Figure 2(A). Induction of 54Mn SOD in aniline-exposed rabbit lung. Gel fraction numbers 6 and 7 correspond to the achromatic zones of mitochondria1 SOD of control (U; 54Mn treated) and exposed (M; 54Mn+aniline treated) animals.

230 - 210-

190-

m

2 5 150 '-i = 110 8 I3O/

3 0 1 0 2 L 6 8 10 12 I& 16

FRACTION N U M B E R

(B) Induction of 54Mn SOD in aniline-exposed rabbit lung. Gel fraction numbers 7 and 8 correspond to the achromatic zones of post-mitochondria1 SOD of control (M, s4Mn treated) and exposed (U; 54Mn+aniline treated) animals.

" O F 170 n

sol 0 2 1 L I 6 1 1 8 10 I 12 I I & 16 I

FRACTION N U M B E R

Figure 3(A). Induction of 65Zn SOD in aniline-exposed rabbit lung. Gel fraction numbers 5 and 6 correspond to achromatic zones of mitochondria1 SOD of control (W; 65Zn treated) and 5, 6 and 7 to exposed (U; 65Zn+aniline treated) animals,

3 0 0 0 2 4 6 I) 10 12 1 A 16

FRACTION N U M B E R

( 8 ) Induction of 65Zn SOD in aniline-exposed rabbit lung. Gel frac- tion numbers 6 and 7 correspond to the achromatic zones of post-mitochondria1 SOD of control (M; 65Zn treated) and 6, 7 and 8 to exposed (M; 65Zn+aniline treated) animals.

strate the specific induction of SOD isoenzymes in response to aniline exposure. The capacity of the tissue to incorporate a metal into an inducible protein has been utilized to demonstrate the induction. The data with 6SZn incorporation show that in addition to induc- tion of a new SOD isozyme, the other compounds also showed an increase. In the case of 54Mn a low increase in the mitochondria1 components is understandable, since mitochondria are also involved in aniline toxicity.

12 P. K A K K A R AND P. N. VISWANATHAN

210r

S O L I I I I I 1 I 0 2 4 6 8 1 0 1 2 1L

FRACTION NUMBER Figure 4(A). Incorporation of 14C amino acids into SOD in control (W; ''C treated) and exposed (0-4; ''CSaniline treated) rabbit lung. Gel fraction numbers 5 and 6 correspond to achro- matic zones of mitochondrial SOD.

200 -

w 5 180- z 1 ,160-

5 1LQ- 0 V

m I-

I 2 O t FRACTION NUMBER

( 6 ) Incorporation of 14C amino acids into SOD in control (U; 14C treated) and exposed (0-4; ''C+aniline treated) rabbit lung. Gel fraction numbers 6 and 7 correspond to achromatic zones of post-mitochondria1 SOD.

The detection of 54Mn in the achromatic zone of the cytosolic component may be due to an exchange with metals of the cytosolic component. In animal tissues, it is believed that mitochondrial SOD is an Mn and the cytosolic one a Cu-Zn containing type.I5 Since no study of isozymic forms is available in lung, the present preliminary findings can only be indicative of the exist- ence of multiple molecular forms of SOD in lungs. As the responses of individual isozymes to aniline vary, a specific regulatory modulation is apparently operating. The new isozymes along with both mitochondrial and cytosolic constitutive enzymes may be formed to defend against the free radicals produced in aniline metabolism. An efficient defensive response against active oxygen in mitochondria and cytosol can be achieved by the induction of SOD in both intracellular compartments. The role of SOD against toxicity induced by a variety of xenobiotics has been reported'h-ls and the present results clearly indicate the modulation of SOD synthesis to meet the stress of aniline exposure. It may be pointed out that, under the same conditions of aniline exposure, aniline hydroxy- lase is induced and mitochondrial 45Ca transport altered along with ultrastructural changes in mitochon- drial and endoplasmic reticulum (unpublished results). Thus biomembranes could be early targets in aniline toxicity and the enhanced SOD level could be a defen- sive adaptation. The interrelation of regulation of SOD and biotransformation of xenobiotics in lung, evident from the present data could be of interest in under- standing the molecular mechanisms in the biochemical toxicology of inhaled xenobiotics.

Acknowledgement

The authors are grateful to Professor P. K. Ray, Director, Industrial Toxicology Research Centre, Lucknow for his interest in this work. This investigation was financed by The Council of Scientific and Industrial Research, New Delhi, India. This manuscript has been ghost written to this presentation by the Technical Editor of this journal, David Clegg.

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SOD ISOZYMES AFTER ANILINE EXPOSURE 13

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