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Protective Role of Ascorbic Acid Against Asbestos Induced Toxicity in Rat Lung: In Vitro Study a

a

a

Sikandar G. Khan , Shahid Ali & Qamar Rahman a

Industrial Toxicology Research Centre, P.Box-80, M.G. Marg, Lucknow, UP, India Published online: 11 Apr 2015.

To cite this article: Sikandar G. Khan, Shahid Ali & Qamar Rahman (1990) Protective Role of Ascorbic Acid Against Asbestos Induced Toxicity in Rat Lung: In Vitro Study, Drug and Chemical Toxicology, 13:2-3, 249-256 To link to this article: http://dx.doi.org/10.3109/01480549009018125

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DRUG AND CHEMICAL TOXICOLOGY, 1 3 ( 2 & 3 ) , 2 4 9 - 2 5 6 ( 1 9 9 0 )

PROTECTIVE ROLE OF

ASCORBIC ACID AGAINST ASBESTOS INDUCED

TOXICITY IN RAT LUNG: IN VITRO STUDY Sikandar G . Khan, Shahid A l i

and Qamar Rahman

Industrial Toxicology Research Centre

Downloaded by [University of Otago] at 23:10 24 July 2015

P.Box - 8 0 , M.G. Marq. Lucknom (U.P.) India

ABSTRACT Asbestos f i b e r s adsorb cytochrome P-450 and P-448 proteins from r a t lung micosomal fractions and liberate heme from cytochrome P-448 o n prolonged incubation i n v i t r o . f u r t h e r , f i b e r s , decrease t h e a c t i v i t i e s of benzo(a)pyrene hydroxylase and g1utathione-Stransferase i n microsomal and cytosolic fractions respectively. Mineral f i b e r s also stjpulate both t h e enzymatic ( NADPH-induced) and non-enzymatic (Fe -induced) l i p i d peroxidation i n microsomal fractions. Preincubation of microsomal and cytosolic fractions with a physiological concentration of ascorbic acid ameliorates, to a large extent, the changes induced by asbestos f i b e r s .

INTRODUCTION In

earlier

studies

(1,2),

we

reported

mineral f i b e r s adsorbed cytochrome P-450 the

l u n g microsomal fractions,

cholanthrene

(3-MC)

incubation i n vitro.

treated

that

and P-448

carcinogenic proteins from

isolated from control and 3-methyl

rats,

and released heme on Drolonqed

These f i b e r s also altered xenobiotic metaboliz-

i n g enzymes and induced enzymatic and non-enzymatic l i p i d peroxi-

dation (LPO) i n v i t r o and i n vivo i n r a t lungs. significantly tissue

&

depleted

w.

the

Ascorbic

lung's

defense

against

tection

against

experimental

level

of

acid,

an

environmental chemical

ascorbic important pollutants

Further, chrysotile acid

in

Dulmonary

component

(3)

carcinogenesis

of

affords

(4).

In

the prothe

present study, effects of ascorbic a c i d , on crocidolite and chryso249 Copyright 0 1990 by Marcel Dekker, Inc.

2 50

KHAN, ALI, AND RAHMAN

tile-induced

biochemical

changes

in

rat

lung,

have,

therefore,

been investigated.

MATERIALS AND METHODS Chrysotile and crocidolite , UICC standard reference

samples

were given a s a gift by D r . J.B. Leinweber, John-Manville M i l l s ,USA. Female 180 gm

were

albino

rats

from

injected

with

3-methylcholanthrene

the

ITRC

colony,

weighing

150-

dissolved

(3-MC)

i n corn o i l ( 2 0 mglkg body w t ) intraperitoneally for four consecutive

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days.

The animals were killed by cervical dislocation, and l u n g

microsomal and cytosolic fractions were separated by t h e procedure

(5).

suggested by Johannesen were

incubated

dust/mg

in

protein)

For adsorption studies,

37OC for

at

the

(0.2

mM).

and

cytochrome P-450

1 hour

presence

with

and

asbestos

absence

of

microsomes

(1 mg

fibers

ascorbic

acid

The contents were centrifuged a t 3000xg for 10 minutes and

P-448

supernatants

in

by t h e method of Omura and Sat0 ( 6 ) .

were

quantitated

For t h e heme release studies,

t h e incubation was done for 72 h r s and 105,OOOxg supernatant used

for

heme

(7).

determination

This time

selection is

was

based

on t h e observation that heme release occurs substantially a t t h i s time interval. ficant. bated

Before t h i s time,

separately

500 p g

with

1 h r with constant s t i r r i n g . at 0.2

at

37OC

for

mM concentration 10 m i n u t e s p r i o r to t h e exposure to dust.

studies,

microsomes

were incubated with sornes,

glutathione-S-transferase

bubbled

with

For

(9)

l i p i d peroxidation

1 0 0 % oxygen

500 p g chrysotile/mg protein.

(1 minute)

In t h e micro-

t h e order of addition of various constituents was t h e same The final concentration of NADPH, iron and

a s given i n Table 3. ascorbic

acid and

0.4

were

incubation

stirring

and

(8),

i n 3000xg supernatants.

determined

(LPO)

The

chrysotile/mg protein

Ascorbic acid was added to t h e system

Benzo(a)pyrene hydroxylase were

t h e release was not very signi-

For enzyme a c t i v i t i e s , microsomes and cytosol were incu-

was LPO

mM,

carried

in

2.5

out

3000xg

at

mM

and

mM

0.2

37OC for 2

supernatant

was

respectively.

h r s with assayed

constant

(10.11).

The selection of t h e dose 1 mg dustlmg protein for t h e adsorption studies and

0.5

mg

dust/mg

protein

in

enzyme

and

is based on our unpublished data where dose-response

LPO

studies

relationship

PROTECTIVE ROLE OF ASCORBIC ACID suggested

the

maximum

251

effects

these

on

doses.

Similarly,

the

rnM ascorbic acid dose is based on our data where

selection of 0 . 2

we studied effect of ascorbic acid i n the concentration range0.05mM1 mM,

but t h e

maximum protection

concentration.

Beyond

could be observed. et a l . ( 1 2 ) .

this

was afforded

concentration

no

only at

further

mM

0.2

protection

Protein was estimated by the method of Lowry

Statistical significance was determined by the Student's

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' t ' t e s t (13) and a value of ~ 0 . 0 5was considered to be significant.

The data i n Table 1 shows that the incubation of l u n g microsomes with asbestos f i b e r s adsorbed cytochrorne P-450 and P-448. Ascorbic

acid

heme-proteins ment the

significantly

.

(P