Chemical and biological effects of oxygen radicals as a consequence of exposure to benzene, and the antioxidant potentials in liver and

s^rum during oxidative stress

A DISSERTATION SUBMITTED'

TO

DEPARTMENT OF BIOCHEMISTRY

( Faculty of Life Sciences )

ALIGARH MUSLIM UNIVERSITY, ALIGARH

For the Degree of

Master of Philosophy IN

Biochemistry

BY

Sarfaraz Ahmad

PETROLEUM TOXICITY DIVISION

INDUSTRIAL TOXICOLOGY RESEARCH CENTRE

LUCKNOW

1995

Dedicated

To

MY RESPECTED PARENTS

With

LOVE & AFFECTION

DS2635

2 7 FEB i m

184^

O.^:?"^EED-2002

9

DEPARTMENT OF BIOCHEMISTRY F A C U L T Y O F L I F E S C I E N C E S

ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA)

TELEPHONE: 400 741

TELEX: 664—230 AMU IN

Ref. No.- Dated 1,4,1995

CERTIFICATE

This i s t o c e r t i f y t h n t t h e work embodied

in t h i s d i s s e r t a t i o n e n t i t l e d •CHEMICAL AND BIOLOGICAL

EFFECTS OF OXYGEN RADICALS AS A CONSEQUENCE OF EXPOSURE

TO BENZENE, AND THE ANTIOXIDANT POTENTIALS IN LIVER

AND SERUM DURING OXIDATIVE STRESS' i s an o r i g i n a l r e s e a r c h

work done by MR. SARFARAZ AHI-IAD unddr my supe i r r i s ion and

i s s u i t a b l e fo r the award of M.Phi l degree in BIOCHEMISTRY,

V/V-w t ^ J - ^ Ipff-y _

( Jawaid I q b a l ) Ph.D.

fndusiria! Toxicology Research Centre

Pax No. — OS22-248227

Telex — 05J5-2456 - f t A ^ MAHATMA GANDHI MAR

f 2 4 0 1 0 7 ^ ^ ^ ^ ^ P^ST nOX No. 30 Telephone | 2 4 185 6 ^ W W W L U C IC N O VV-226001

Iclcgrnm"! N T O X r - ^ i a ^ " ^ ^ . ^ U.P . INDIA

\Li, (. OlvRUSI'ONDl N( U SIKJUI U UU \ D l ) R r S 3 e o lO DIKHCIUR

Dr. G.S. Rao Assistant Director Petroleum Toxicology Division

C E R T I F I C A T E

This is to certify that the work embodied in this dissertation entitled "Chemical and biological effects of oxygen radicals as a consequence of exposure to benzene, and the antioxidant potentials in liver and serum during oxidative stress" has been carried out by Mr. Sarfaraz Ahmad under my supervission.

He has fulfilled the requirements of the Aligarh Muslim University, Aligarh for the degree of Master of Philosophy in Biochemistry.

This work included in this dissertation is original unless stated otherwise and has not been submitted for any other degree.

Dated: ^ ^ ^ ' '^ ^^ [ DR. G.S. RAO ]

A C K N O W L E D G E M E N T S

I t gives me immense pleasure to record my deep sense of

gratitude and indebtedness to my supervisor Dr. G.S. Rao,

Assistant Director, Petroleum Toxicity, Industrial Toxicology

Research Centre, Lucknow whose unvaluable guidance, incessant

encouragement and affectionate behaviour brought my research

efforts to the completion of this dissertation.

It is my pleasure to express my thanks to Dr. Javed Iqbal,

Department of Biochemdstry, Aligarh Muslim University, Aligarh

for his keen interest, constant encouragement and support.

I wish to express my sincere thanks to Prof. S.M. Hadi,

Chairman, Department of Biochemistry, Faculty of Life Sciences,

A.M.U. , Aligarh for his interest and help rendered during the

tenure of this work.

I am indebted to Dr. R.C. Srimal, Director, Industrial

Toxicology Research Centre, Lucknow for providing me laboratory

facilities.

Well, I can't forgive myself if I fail thanking to Dr.

G.S.D. Gupta, Dr. S.K. Goel, Mr. Ram Surat and Mr. Abdul Aziz

for their active support and amiable cooperation.

I record my sincere thanks to Prof. A.M. Siddiqui, Prof.

M. Saleemuddin, Dr. A.N.K. Yusufi, Dr. Riaz Mehmood and Dr.

Qayyum Husain, who revealed me the fascinating field of

Biochemistry.

I also acknowledge the stimulating companionship and

valuable cooperation of all my colleagues especially Dr.

Navedul, Shoeb, Farrukh, Vaqar, Shakeel and Asif.

Words fail to express my deep sense of love and respect to

my parents, brothers and sister for extending their moral

support and maximum cooperation without which dream of my

writing this dissertation would not have materialized. I also

acknowledge love and affection to my all relatives and well

wishers.

Financial assistance from the Indian Council of Medical

Research, New Delhi is gratefully acknowledged.

And, at last but nevertheless not the least I thank Mr.

Lakshmi Kant Shukla for typing this dissertation neatly and in

time.

D a t e : SARFARAZ AH24AD

C O N T E N T S

P a g e No .

P r e f a c e

Abbreviations

List of Tables

List of Figures

Introduct ion

Review of Literature

Aims and Objectives

Materials and Methods

Results

Discussion

Bibliography

I

III

IV

V

1 - 12

13 - 19

20 - 21

22 - 34

35 - 53

54 - 64

65 - 73

Preface

Benzene is a well known xenobiotic environmental pollutant

and is a natural constituent of crude oil. Emissions from

burning coal and oil, benzene waste and storage operations,

motor vehicle exhaust, evaporation from gasoline service

stations and use of industrial solvents increase benzene level

in the air. Since tobacco contains high level of benzene,

tobacco smoke is another source of benzene in air. People

living around hazardous waste sites, petroleum refining

operations, petrochemical manufacturing sites, or gas station

may be exposed to higher levels of benzene in air.

Benzene causes disorders in the blood. People who breath

benzene for long periods may experience harmful effects in the

tissues that form blood cells, especially the bone marrow.

These effects can disrupt normal blood production and cause a

decrease in important blood components. A decrease in the

number of functional red blood cells can lead to anemia. Long

term exposures to high levels of benzene in the air can cause

cancer of the tissues that forn . white blood cell leukeria) .

II

the cell to defend itself against the oxygen derived species

resulting in oxidative injury. Benzene exposure has been shown

to generate organic free radicals and superoxide radical and

lead to accumulation of iron.

In view of the protective role of antioxidants, it might

be of interest to evaluate the antioxidant activities in serum

after exposure of benzene to experimental animals (viz. uric

acid, albumin, ascorbic acid, oC -tocopherol, ceruloplasmin,

ferroxidase activity and catalase) and to evaluate the

transition metal ion concentrations (viz. iron and copper) in

liver and in isolated rat liver nuclei by atomic absorption

spectrophotometery. In view of accumulation of £-aminolevulinic

acid (ALA) and increased excretion of ALA in urine during

benzene exposure it is worthwhile to study autooxidation of ALA

and the ability of oxygen radicals thus produced has the

capacity to degrade biomolecules. Also to investigate the

effect of copper catalysed autooxidation of ALA to generate

oxyradicals may have physiological significance because trace

amounts of copper were detected in certain extracellular fluids.

A B B R E V I A T I O N S

III

ALA

BSA

b.w.

DNA

RNA

dl

DOC

DTNB

EDTA

g

i.p.

mg

ug

MDA

mM

umole

nmole

PBG

ROS

s.c.

SE

SOD

TEA

TEAR

TCA

w/v

£ -Aminolevulinic acid

Bovine serum albumin

body weight

Deoxyribonucleic acid

Ribonucleic acid

Decilitre

Deoxycholic acid

Dithio-2-nitrobenzoic acid

Ethelene diamine tetraacetic acid

Gram

Intraperitoneal

Milligram

Microgram

Malonaldehyde

Millimolar

Micromole

Nanomole

Porphobilinogen

Reactive oxygen species

Subcutaneous

Standard error

Superoxide dismutase

Thiobarbituric acid

Thiobarbituric acid reactive products

Trichloroacetic acid

Weight by volume

IV

LIST OF TABLES

Si. Page No, No.

Effect of benzene administration on serum 3 concentration of natural antioxidants (Uric acid & Albumin) of albino rats.

2. Effect of benzene administration on hepatic free -SH content and rate of lipid peroxi­dation of albino rats.

3. Effect of benzene administration on serum ceruloplasmin, liver ascorbic acid concen­tration and liver catalase activity of albino rats.

4. Effect of benzene administration on liver and serum oC-tocopherol concentration of albino rats.

5. Effect of benzene administration on liver -aminolevulinic acid dehydratase activity of male albino rats.

6. Effect of benzene administration on serum ferroxidase activity and iron binding capacity of female albino rats.

7. Effect of benzene administration on total iron and copper metal contents in liver tissue and isolated liver nuclei of albino rats.

8. Formation of TEAR from glutamate or deoxyuridine by £ -aminolevulinic acid (ALA) in the presence of copper ions.

37

39

40

41

42

43

45

9. ALA concentration dependent utilization 5; of ALA and formaldehyde formation from dimethyl sulfoxide during autooxidation of ALA.

10. Release of TEAR from glutamate or deoxy- 5 2 uridine during autooxidation of ALA in the presence of copper and the inhibition by c::ygen radical cc : L, d / e . ^ T O -/~ Q

LIST OF FIGURES

SI. Page No. No.

1. Metabolic pathway of benzene 4

2. Linearity of TEAR formation from 45 glutamate as a function of time during the autooxidation of ALA.

ALA concentration-dependent increase 47 of TEAR formation from glutamate (< and deoxyuridine ( 0 0 ) .

4. Copper ion concentration-dependent 48-49 increase of TEAR formation from (A) glutamate and (B) deoxyuridine.

5. Oxygen consumption with respect to 5 0 increase concentration of ALA.

Introduction

Benzene

Benzene is an aromatic hydrocarbon (CgHg) and is

colourless liquid with a sweet odor. Due to high vapour

pressure, benzene evaporates into air very quickly and is highly

flammable. It is abundant in coal tar distillates and is a by

product of coke and petroleum refining. Natural sources, which

include volcanoes and forest fires, account for a small amount

of benzene in the environment. Today benzene is commercially

recovered from both coal and petroleum sources. Small

quantities of benzene are also derived from coal in the light

oil produced during coke manufacture (Greek, 1990) .

Uses of Benzene :-

In the past benzene was widely used as a solvent, but this

use is now decreasing due to its toxic nature. The T.ajor uses

of benzene are ir th3 production of ethylbenzene, zimene and

cyclohexane. Otner industrial use of rr.e benzene 13 in the

production cf rutoer plastics, paints, adr.esives, nylrr. fibres,

artificial leather and chemicals (such as maleic anhydride and

aniline) (Eveleth, 1990). Benzene is also a component of

gasoline since it occurs naturally in crude oil and since it is

a by product of oil refining processes. Benzene is especially

important for unleaded gasoline because of its anti-knock

characteristics. For this reason, the concentration of

aromatics such as benzene in unleaded fuels has increased (Brief

et al., 1980).

General Population and Occupational Exposure :-

Virtually all (99.9%) of the benzene released into the

environment is emitted into the air. Inhalation is the dominant

pathway of human exposure, accounting for more than 99% of the

total daily intake of benzene (Hattemer-Frey et al., 1990). The

general population is exposed to benzene mainly through

inhalation of contaminated air particularly in areas of heavy

motor traffic and around gas stations and through tobacco smoke

from both active and passive smoke. Since benzene is a

constituent of auto exhaust, people who spend more time in cars

or in areas of heavy traffic will have increased personal

exposure to benzene. Pumping gasoline can also be a significant

source of exposure. A study reported a breathing - level

benzene concentration of 1 ppm while pumping gas (Bond et al.,

1986) .

Probably the most significant source of benzene exposure

for the general population is active smoking of tobacco.

Smoking accounts for about half of the total population burden

of exposure to benzene (Wallace, 1989). All outdoor source.

3

including automobile exhaust and stationary source emissions,

account for only about 20% of the total population exposure to

benzene (Wallace, 1989) .

Other source of inhalation exposure to benzene include air

around hazardous waste sites, industrial facilities and

emissions from consumer products (paints, adhesives, rubber

products and tapes).

Benzene metabolism :-

Benzene requires metabolism to induce its toxic effects

and follows similar pathways in humans and animals. The

metabolism of benzene occurs in bone marrow also, as well as in

the liver and is metabolized by cytochrome P-450 dependent

mixed-function oxidase enzymes. Metabolism of benzene is very

complex (Figure- 1) and is primarily metabolized to phenol,

catechol, hydroquinone, 1,2,4-benzenetriol, trans, trans-muconic

acid and benzene dihydrodiol. The majority of these phenolic

metabolites are excreted in urine as glucuronide and sulphate

conjugates. In experiments with rabbits, accumulation of

several polyphenolic metabolites of benzene have been observed

in bone marrow against the concentration gradient (Greenlee et

al., 1981).

Initially benzene is metabolized to benzene oxide (Jerina

et al., 1968), which is further metabolized by one of four

pathways. The first metabolite is formed by rearrangement of

the benzene oxide to phenol spontaneously. Phenol is either

conjugated to glucuronide and sulphate directly or is oxidized

to hydroquinone and followed by conjugation to hydroquinone

00

E o

a o <

0) c fl) N C 4)

J3

"4-1

o

? SI •*->

a

o X! +-•

:2

bs

glucuronide or sulphate. The second pathway for benzene oxide

metabolism consisted of reaction with glutathione and subsequent

modification of the mercapturic acid (Henderson et al. , 1989).

The third pathway for benzene metabolism involved in conversion

of benzene oxide into benzene dihydrodiol by epoxide hydrase

(Tunek et al. , 1981). Benzene dihydrodiol is converted into

catechol by oxidation of benzene dihydrodiol by a soluble NADP" -

dependent enzyme, benzene dihydrodiol dehydrogenase (Ayengar et

al., 1959) and finally conjugation to glucuronide and sulphate.

Benzene dihydrodiol is also converted into 1,2,4-benzenetriol by

some unknown mechanism, which is one of the potent toxic

metabolite of benzene. The fourth pathway of benzene oxide

metabolism consists of the ring opening reaction resulting in

the formation of trans, trans-muconic acid. However the

metabolic pathway of trans, trans-muconic acid in vivo is yet to

be identified (Latriano et al., 1986). The corresponding

dialdehyde, trans, trans-muconaldehyde has been shown to be a

precursor of muconic acid in vivo (Witz et al., 1990).

Benzene Toxicity :-

Benzene exposure leads to the development of bone marrow

depression characterized by progressive leucopenia, anemia and

pancytopenia (Snyder, 1987) . The responses to chronic benzene

exposure have been classified as either bone marrow depression

and aplastic anemia or the generation of acute myeloblastic

leukemia (AMD and related forms of acute lymphocytic/ncn-

lymphocytic leukemia. Collectively, these abnorm.alities have

been termed as preleukemic syndrome or myeicdysplasric syndrome

(Bagby, 1986; Tricot, 1991) . Irons (1988) and Tricot (1991)

suggested that the spectrum of bone marrow toxicities produced

by exposure to benzene represented a form of myelodysplastic

syndrome resulted in bone marrow depression and that might lead

to fatal aplastic anemia. Anon (1982) reported that benzene is

a human leukemogen. Chronic benzene poisoning exhibited a

variety of changes in the number of basophils, eosinophils,

lymphocytes, and other bone marrow function in workers (Aksoy et

al., 1971).

Goldstein et al., (1982) reported on the production of a

limited number of myelogenous leukemias in rats and mice exposed

to benzene by inhalation. The reports by Maltoni et al. (1989)

and Huff et al., (1989) of benzene-induced carcinogenicity in

rats and mice given benzene orally focussed on solid tumors and

multiple carcinogenic foci. The zymbal gland was a primary

carcinogenic site in both sexes of both species. Later studies

using the inhalation route (Maltoni et al., 1989) also

demonstrated the multipotential carcinogenic activity of

benzene. Excluding the studies by Yin et al., (1987) there is

no strong evidence for benzene-induced solid tumors of these

types in human. Snyder et al., (1980) also reported the

production of thymic lymphoma after inhalation of benzene in

C57B1/6J mice but not in AKR mice. Farris et al., (1993)

observed the malignant lymphoma along with preputial gland

carcinomas and lung adenomas in CBA/Ca mice exposed to benzene

through inhalation. Neither the Cronkite et al. , ;i989' r.cr the

Farris et al., (1993) studies demonstrated acute granulccytic

leukemia in benzene inhaled mice.

Snyder and Chatterjee (1991) and Yardley-Jones (1991) have

studied the pathway of benzene metabolism and the role of

benzene metabolites in the production of benzene toxicity.

Hepatic metabolism of benzene appears to be a critical step in

the mechanism of benzene toxicity. Lee ez al. , (1981) reported

that early erythroblasts are sensitive to benzene and its

metabolites. Benzene has long been known to produce chromosomal

abnormalities (Eastmond, 1993) and suggested that benzene may

have caused chromosom.e breakages and rearrangements in a stem

cell that proliferated, leading to erythroleukemia. Eastmond

(1993) also observed that the quinone mietabolites of benzene can

cause aneuploidy to chromosome nondisjunction and nonrandom

chromosomal alteration in individuals with advanced forms of

benzene-induced myelotoxicity. The association between

chromosomal abnormalities and leukemia in benzene exposed rats

has been reviewed by Le Beau and Larson (1991). Tice et al.,

(1980) exposed mice to benzene by inhalation and demonstrated an

increased frequency of sister chromatid exchanges in bone marrow

cells and suggested that benzene metabolites were responsible

for these effects. Muconaldehyde, a hypothesized ring open

benzene metabolite also produced sister chromatid exchanges in

B6C3F1 mice (Witz et al., 1990).

The highly electrophilic metabolites such as p-

benzoquinone and others are generated during benzene exposure in

experimental animals and form covalently bound adducts with both

nuclear and mitochondrial DN7A (Zastrcr.i 1?S3) . Kclacnar.a ez

al., (1993) have demonstrated that benzene and its phenolic

metabolites; phenol, hydroquinone and 1 I 4-benzenetrioi, induce

8

oxidative damage in the genome in the form of 8-

hydroxydeoxyguanosine in HL-50, promyelocytic leukemic cells and

in the bone marrow of mice. Cheng et al. , (1992) demonstrated

that benzene induced active oxygen radical production resulted

in 8-hydroxydeoxyguanosine formation in vivo which may play a

role in genotoxicity and leukemogenesis. Formation of 8-

hydroxydeoxyguanosine adduct is known to cause G-T and A-C base

substitutions.

Earlier reports have shown that benzene itself is probably

not the actual toxicant, but is converted by hepatic metabolism

to one or more metabolites and accumulated against the

concentration gradient in bone marrow and exerts its toxic

effects (Smith et al., 1989). Recent studies involve the

polyphenolic metabolites as the toxic intermediates due to their

capability to undergo autooxidation (Rao and Pandya. 1989).

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are the

two principal enzymes in bone marrow which are responsible for

the formation of quinone and semiquinone free radicals and

activation of oxygen to superoxide radicals. These free radical

metabolites of benzene with dioxygen and cellular targets such

as DNA, GSH, protein and lipid are responsible for the benzene-

induced myelotoxicity and leukemia (Subrahmanyam et al., 1991a).

Legathe et al., (1994) have demonstrated pharmacokinetic

interaction between benzene metabolites, phenol and

hydroquinone, in B6C3F1 mice. The consequence of which may be a

higher o'jantity of 1,4-benzoquinone formation from hydroquinone.

Many investigators have shown that benzer.e exposure interferes

with iron metabolism and heme biosynthesis in experimental

animals. These studies include decreased incorporation of ^Fe

into circulating erythrocytes (Lee et al., 1974), inhibition of

heme biosynthesis (Freedman et al., 1977; Rao and Pandya, 1980)

and depletion of the regulatory heme pool (Siddiqui et al. ,

1988) . Existence of an intracellular pool of low molpcular

weight iron compounds, maintaining a dynamic equilibrium between

iron uptake, iron storage and iron incorporation into its final

biochemical form have been described (Jacobs, 1977). Benzene

administration led to accumulation of iron in bone marrow cells

which can catalyse the formation of aldehydic products (base

propenal) from DNA in presence of bleomycin (Rao et al., 1990).

In all probability benzene alters heme metabolism of erythroid

cells resulting in the increased internalization of iron

transferrin-receptor complex and uptake of internalized iron is

almost entirely restricted to erythroblasts. Recently in an in

vitro study, Rao (1991) observed that hemin catalyzed the

autooxidation of hydroquinone or 1,2,4-benzenetriol in the

presence of reducing agent and resulted in the formation of

aldehydic products from glutamate, deoxyuridine or DNA through

the formation of reactive oxygen species. Hemin is essential

for the erythroid differentiation process and accumulates in the

immature red blood cells in nanomolar quantities as these cells

differentiate (Lo et al., 1981). In immature red blod cells in

bone marrow it has been noted that heme accumulates in the

nucleus as these cells differentiate and nicking of nucelar DNA

has been reported as it binds covalently to both protein and DNA

(Mager and Bernstein, 1979; Lo et al., 1981). It has been

postulated tr.at this action of hemin might account for its

10

influence in the differentiation of these and other cell types

(Chen and London, 1981) . In view of the accumulation of

polyphenolic metabolites after exposure to benzene and the

presence of excessive amounts of heme in bone marrow cells offer

an alternative mechanism for the bone marrow depressant effect

of benzene (Rao, 1991).

Antioxidants & its Importance :-

The formation of highly reactive oxygen containing

molecular species is a normal consequence of a variety of

essential biochemical reactions. Endogenous source of free

radical formation include those that are generated and act

intracellularly as well as those that are formed within the cell

and are released into the surrounding area. Oxidases and

electron transport systems are prime, continuous sources of

intracellular reactive oxygenated free radicals. These reactive

free radicals have capability to cause cellular damage (Machlin

and Bendich, 1987). Exogenous sources of free radicals include

certain pollutants (like benzene), smoke, hyperoxic environments

and pesticides and are metabolized to free radical intermediate

products that have been shown to cause oxidative damage to the

target tissues (Halliwell and Gutteridge, 1985) . Thus aerobic

cells are equipped with an array of antioxidant defences such as

uric acid,_ albumin, oC -tocopherol, free -SH groups and

ferroxidase activity which terminate the propagation of damage

by free radicals.

There are both enzymes and small molecular weignt

molecules with antioxidant caoabilities that can protect acainst

11

the adverse effect of free radical reactions. Antioxidant is

defined as any substance that when present at low concentration

compared to those of an oxidizable substrate, significantly

delays or inhibits oxidation of that substrate (Halliwell and

Gutteridge, 1990) . Antioxidants could act against lipid

peroxidation by decreasing localized O2 concentration and are

preventing the initiation of peroxidation by scavenging species

capable to abstract hydrogen atoms, such as hydroxyl radical

('OH). Antioxidants are also capable of quenching or scavenging

the singlet oxygen which can react directly with membrane lipids

to produce peroxides. Lipid peroxidation is also scavenged by

binding the antioxidants to metal ions in forms that will not

generate reactive species and will not decompose lipid peroxides

to peroxyl and alkoxyl radicals (Halliwell and Gutteridge,

1990) .

Antioxidants are following of two types :- (1) Enzymatic

and (2) Non-enzymatic. Enzymatic antioxidants include

superoxide dismutase (SOD), catalase and glutathione dependent

systems. SOD scavenges the superoxide radicals and generate

hydrogen peroxide (H2O2) which is less reactive than superoxide

radical. Catalase is a heme protein, which catalyzes the

reaction to convert H2O2 into water and O2. Glutathione

dependent systems scavenge the hydroxyl radical on the expense

of thiol group and play a vital defence against lipid

peroxidation and are major interceptor of chemical

mutagenesis/carcinogenesis (Chasseaud, 1979) . The in-.pcrtant

non-enzymatic antioxidants are uric acid, albumin, oC -

tocopherol, ascorbic acid and ceruloplasnin. Uric acid has the

12

ability to scavenge oxidizing species such as singlet O2, HOCl

and peroxyl radicals and inhibits lipid peroxidation. Uric acid

can act as an antioxidant, both by binding iron and copper ions

in forms that do not accelerate free radical reactions and by

directly scavenging the free radicals (Ames et al. , 1981).

Albumin can bind free Cu "*" ions and site directs any radical

damage onto the albumin molecule itself and is powerful

scavenger of HOCl transports bilirubin (Hartman and Shankel,

1990) . oC -Tocopherol is most important lipid soluble chain

breaking antioxidant and breaks chains by trapping peroxyl and

alkoxyl radicals. Ascorbic acid has the ability to regenerate

oC-tocopherol by reducing oC -tocopheryl radicals at the surface

of membranes and is most important plasma antioxidant which

scavenges water soluble peroxyl (RO2) radicals (Hartman and

Shankel, 1990). Ceruloplasmin also inhibits lipid peroxidation

stimulated by iron and copper ions and inhibits iron stimulated

hydroxyl radical generation. It reacts slowly with superoxide

and hydrogen peroxide. Ferroxidase activity does not result in

formation of damaging oxygen radicals, unlike the non-enzymatic

oxidation of Fe , which gives 'OH and other powerful oxidants

(Halliwell and Gutteridge, 1990) .

Review of Literature

13

Benzene is a known haeinatotoxic and leukemogenic agent

(Snyder et al., 1977) and chronic exposure to concentrations

ranging from 25 to 1000 ppm results in progressive degeneration

of the haematopoietic system resulting in pancytopenia in

experimental animals (Gerarde and Ahistrom, 1966). Occupational

exposure to benzene has been associated with various blood

dyscrasias and a variety of leukemia in humans (Rinsky et al.,

1987; Aksoy, 1989).

Accumulating evidence indicate that the expression of

benzene toxicity requires metabolism of the parent compound to

one or more toxic species (Sammett et al., 1979). Several

metabolites including phenol, catechol, hydroquinone, 1,2,4-

benzenetriol, trans, trans-muconic acid and benzene dihydrodiol

have been identified in the urine of rabbits exposed to •'C

benzene (Parke and Williams, 1953) . Inspite of the fact that

the metabolism of benzene is extensively investigated and well

understood, the intermediate agent(sj resDonsible for

14

haematotoxicity has not been identified. Moreover, the relation

of benzene metabolism to leukemogenesis may very well differ

from that usually observed in polycyclic aromatic hydrocarbon

induced carcinogenesis.

Many reports showed that benzene metabolism occurs in bone

marrow also, as well as in the liver. Recent studies involve

the polyphenolic metabolites as the toxic intermediates due to

their capability to undergo autooxidation (Rao and Pandya,

1989). Andrews et al., (1979) have shown the presence of

cytochrome C reductase in bone marrow and demonstrated in vitro

formation of hydroquinone, catechol and an unidentified

metabolite in microsomes isolated from bone marrow incubated

with benzene. Snyder et al., (1978) observed that benzene

toxicity in mouse is associated with accumulation by bone marrow

of labelled benzene and to a greater extent of its metabolites.

Above evidences suggest that a reactive metabolite of benzene

may be the ultimate haematotoxic agent.

Polyphenolic metabolites of benzene- have been identified

in rabbits exposed to benzene and accumulation of polyphenols in

bone marrow against the concentration gradient has been reported

(Irons, 1985). Metabolites of benzene have been reported to

cause alterations in bone marrow (Morimoto and Wolff, 1980) and

hydroquinone and 1,2,4-benzenetriol the two principal

metabolites of benzene have been shown to be toxic and the

suggested mechanisms include free radical formation via,

superoxide and the cc"alent oirAmc of the semiquinones zc ^NA,

RNA and other csllular macrcroiecules (Tunek ez al. , 153:,-

Irons, 1985). Involven"ent: cf ac::ive cxvcen radicals v,a sr.z;:z

in the cytotoxic effect of polyphenolic metabolites of benzene

besides the semiquinones which can bind to macromolecules (Rao

and Pandya, 1989) .

Quinone and semiquinone metabolites of polyphenols,

through their capacity to react with nucleophilic groups of

amino acids are reported to inactivate critical enzymes like DNA

polymerase (Graham et al. , 1973), RNA polymerase II (Nagaraja

and Shaw, 1982) and reverse transcriptase (Wick and Fitzgerald,

1981) . Quinone or semiquinone metabolites of hydroquinone and

1,2,4-benzenetriol have been shown to inhibit mRNA synthesis

(Kalf et al., 1982), microtubule polymerization (Irons and

Neptun, 1980), mitogen stimulation of lymphocyte growth (Weirda

and Irons, 1982), effect macrophage function and activation

(Lewis et al., 1988a) and form adducts with DNA (Jowa et al.,

1986). Autooxidation of polyphenolic compounds in presence of

low concentrations of transition metal ion copper resulted in an

increased production of active oxygen species which can release

aldehydic products from glutamate or DNA (Rao and Pandya, 1989).

Hydroquinone and catechol have been shown to be much more

toxic to bone marrow cell cultures than benzene (Parmentier and

Dustin, 1953). The covalent binding of hydroquinone and

catechol in the bone marrow and subsequent potential pathways

for the activation to benzoquinone and 1,2,4-benzenetriol which

readily autooxidize to respective semiq jinone and/or quinones

may play a significant role in the cytoccxicity of benzene. The

quinone metabolites may arise from a aireci accivauion mechanism

in bone marrow by superoxide radicals (Hansen et al., 1978) .

16

Considerable research has focused on the interaction of

benzene and its metabolites with DNA. Initial studies by Lutz

and Schlatter (1977) recovered radioactivity bound to rat liver

DNA following the administration of tritiated and C-benzene to

male rats. Additional studies by Gill and Ahmed (1981) and

Arfellini et al., (1985) demonstrated that the benzene or

metabolites were capable of binding to bone marrow DNA following

in vivo administration. More recently, a series of

investigations have focussed on the ability of the individual

benzene metabolites to bind to deoxynucleotides and DNA in vitro

(Rushmore et al., 1984; Jowa et al., 1986; Snyder et al., 1987;

Bauer and Snyder, 1989; Latriano et al., 1989; Pongracz et al. ,

1990). These studies have indicated that 1,4-benzoquinone,

hydroquinone, phenol, 1,2,4-benzenetriol, trans, trans-

muconaldehyde, catechol are all capable of forming adducts with

deoxynucleotides and DNA during in vitro incubations. However,

the nature of the binding in the bone marrow and the identity of

the reactive species involved remain largely unknown.

One consistent observation that has been made in benzene

exposed humans and animals is the appearance of structural and

numerical chromosomal aberrations and sister chromatid exchanges

in lymphocytes and bone marrow cells indicating that benzene is

genotoxic (Forni et al., 1971; Morimoto and Wolff, 1980; Tice et

al., 1980; Dean, 1985; Yardley-Jones et al. , 1990). Chromosome

and chromatid deletions, gaps, chromatid exchanges,

hyperdiploidy and micronuclei induction, all have been detected

to some extent. Since the induction of chromosomal aberrations

in humans have been associated with a variety of neoplastic

17

development (Hecht and Hecht, 1987; Sandberg, 1987), these

chromosomal aberrations could also contribute to the myelotoxic

and leukemogenic effects of benzene in humans. Benzene exposure

to experimental animals has been shown to inhibit ENA, RNA and

protein synthesis (Falf, 1987; Cooper and Snyder, 1988).

Benzene metabolites interfere with DNA replication and

transcription in rat liver and bone marrow mitochondria in

vitro, that has been attributed to the inhibicicn cf DNA

polymerase gamma activity by hydroquinone and 1,4-benzoquinone.

Studies by Lee et al. , (1988) have confirmed that an inhibition

of DNA synthesis occurs in bone marrow following the in vivo

administration of 1,2,4-benzenetriol to mice and have indicated

that inhibition occurred at doses that had no inhibitory effect

on the synthesis of protein or heme. These authors reported

that benzene itself had the ability to inhibit DNA synthesis in

vitro in cell free incubations following the addition of

benzene.

Previous studies have suggested the involvement: of iron or

copper in benzene metabolite induced DNA damage through a

mechanism involving the generation of reactive oxygen species

(Lewis et al., 1988b; Rao and Pandya, 1989; Kawanishi et al. ,

1989). In vitro studies by Rahman et al., (1989) and Ahmad et

al., (1992) have shown that naturally occurring flavonoid,

quercetin, in the presence of Cu(II) and molecular oxygen caused

breakage of calf thymus DNA, supercoiled pBR 322 plasmid ENA and

single stranded M13 phage DNA. Copper is distribuned throughout

the body with the liver and bone marrov; being rwc -ra or copper

storage organs (Goyer, 1986; Linder, 1991). Copper has been

identified as the essential redox-active centre in a variety of

metalloproteins such as ceruloplasmin, Cu-Zn SOD, cytochrome

oxidase, dopamine E-hydroxylase, tyrosinase, lysyloxidase and

ascorbate oxidase (Linder, 1991). Since copper also exists in

the nucleus and is closely associated with chromosomes and DNA

(particularly Guanine) the chemical-metal redox system might be

responsible for strand breaks in vivo during benzene exposure.

In vitro, am.ong hydroquinone, 1,2,4-benzenetriol, catechol and

phenol; hydroquinone/Cu and 1,2,4-benzenetriol/Cu were the

two efficient DNA cleaving systems (Li and Trush, 1993a).

Recently it was observed that Cu(II) strongly induces the

oxidation of hydroquinone as such may be factor involved in the

oxidative action and toxic to primary bone marrow stromal cells

of DBA/2J mice (Li and Trush, 1993b). They further observed that

Cu(II) is capable of directly reacting with hydroquinone,

causing the one electron oxidation of hydroquinone to

semiquinone radical (SQ') and reactive oxygen species generated

in chemical metal redox system were responsible for in vitro

plasmid DNA damage (Li et al. , 1995). The oxidative activation

of hydroquinone to the electrophilic 1,4-ben2oquinone through a

peroxidase/H202 system has been suggested to play an important

role in the hydroquinone-induced bone marrow target cell injury

(Smith et al., 1989).

It is generally accepted that benzene produces bone narrow

toxicity resulting in pancytopenia in man and l^czr:z.zorY ar.iTials

and at least in man results in aplastic anemia and acute

myeloblastic leukemia (Kalf, 1987). Earlier studies showed that

19

benzene inhibits heme biosynthesis at the level cf £ -

aminolevulinic acid (ALA) dehydratase (Rao and Pandya, 19S0) to

result in ALA accumulation and its loss in urine (Rao ez al. ,

1919); depletes the regulatory heme content (Siddiqui ez al.,

1988), leading to accumulation of iron .Pandya et al. , 1590).

Majority of intracellular iron is stored within the central core

of ferritin, a large multisubunit protein found predominantly in

the liver, spleen and bone marrow (Harrison, 1977. . The

concentration of free or low molecular weight for~3 are

negligible or non existent within the normal cells Rcmslo,

1983). However, superoxide dependent reduccive release cf iron

from ferritin may increase the low molecular weight ircn pool

capable of undergoing redox reactions leading to the fcrniation

of other toxic oxidant radicals. Existense of intracellular

pool of low molecular weight iron compounds maintaining a

dynamic equilibrium between iron uptake, iron storage and iron

incorporation into its final biochemical form have been

described (Jacobs, 1977). Many studies have shown that

inhibition of iron uptake and heme biosynthesis are the

causative factors of benzene induced aplastic anemia Lee et

al., 1974). In all probability benzene alters heme me-abolism

in erythroid cells resulting in the increased internalizarion of

iron transferrin receptor complex. Benzene may also likely

affect by an unknown mechanism, the utilization of internalized

iron, the uptake of which is almost entirely restricted to

erythrcblasts (laccpetta and Morgan, 15=3) resulting ^n the

accumulation of low molecular weight ircn components.

Aims and Objectives

20

A large number of xenobiotics including environmental

agents and therapeutic formulations exert their toxic effects

via radicals which react readily with lipids, nucleic acids,

carbohydrates and proteins and inhibit sulphahydryl dependent

enzymes of the intermediary metabolism. Hydroxy metabolites of

benzene such as hydroquinone and 1,2,4-benzenetriol are

intermediate products of benzene metabolism. These compounds

have been shown to be toxic by the mechanism include free

radical formation via superoxide and covalent binding of the

semiquinones to DNA, RNA and other cellular macromolecules.

Active oxygen species capable of initiating lipid peroxidation

and other deleterious oxidative processes are generally proposed

to result from reactions between oxygen radical and transition

metals. Xenobiotics that lower cellular antioxidants, both

enzynatically and nonenzymatically cause cell injury due to

facilitated attacK on essential cell constituents. This leads

to oxidative stress which has been defined as the inacilitv cf

21

Exposure to benzene requires metabolism to induce its

toxic effects and follows similar pathways in humans and

animals. Inside the body, the metabolites of benzene are

responsible in the formation of reactive oxygen species which

may play a role in benzene induced toxicity. To over come the

toxic effect, the mammalian cells are equipped with an array of

antioxidant defences. During our study the antioxidants level

in serum and liver were evaluated in benzene exposed

experimental rats and this study may provide the clinical and

therapeutic information on biochemical and haematological effect

of benzene.

Materials and Methods

22

Chemicals : -

Benzene of high purity was obtained from Glaxo

Laboratories India (ANLAR). Bathophenanthroline sulfonic acid,

catalase (bovine liver, 2,000-5,000 units/mg protein),

superoxide dismutase (bovine erythrocytes, 2,500-5,000 units/mg

protein), bovine serum albumin, L-glutamate monosodium, S -

aminolevulinic acid, 2-deoxyuridine, oC-tocopherol ^nd dithio-2-

nitrobenzoic acid were obtained from Sigma Chemical Company, St.

Louis, U.S.A. Folin-Ciocalteu's phenol reagent, tris-

hydroxymethyl amino methane, glutathione and trichloroacetic

acid were obtained from Sisco Laboratories, India. 2-

Thiobarbituric acid was obtained from BDH chemicals Limited,

England. All other chemicals were either obtained from C.S.I.R.

Centre for Biochemicals, New Delhi, India or were of analytical

grade.

IncrruiTients :-

Spectronic 21, Bausch and LcrJD, Clark elecnrode assembly

or. a Gilson Medical Electronic Cxygraph, Ultra-turrax T25

23

n o m o g e n x z e x , >iiuxnL:u-Duwiuciii optso-ux opxiuLUi. xaiuiut; Lex , A t o m i c

absorption spectrophotometer, Mettler balance, Dig i ta l pH meter

and Centrifuge (REMI) were the major instruments used in t h i s

study.

Animals :-

Albino rats of either sex (Swiss wistar strain, 100-110

gra) bred at the animal house unit of I.T.R.C. were divided into

six separate groups (three groups male and three groups female)

of six animal each. One group from each sex received benzene

(0.5 ml/kg body weight) intraperitoneally and another group from

each sex received benzene (1.0 ml/kg body weight) subcutaneously

daily for ten days. After the termination of the treatment,

blood was collected into clean tubes from jugular vein and kept

at 4°C. Control and experimental rats were sacrificed by

cervical dislocation and liver and brain were immediately

excised and 10% liver homogenate was prepared in 0.15 M KCl to

study lipid peroxidation. Liver homogenate was also made in

0.02 M ethylenediamine tetraacetic acid (EDTA) for the

estimation of sulphahydryl content, ascorbic acid, oC-tocopherol

and catalase activity. Brain homogenate 2% was also made in

0.15 M KCl for studying ferroxidase activity.

Protein Estimation :-

Protein in different tissues was estimated by the method

of Lowry et al., (1957). Tissue homogenate was precipitated

with trichloroacetic acid (TCA). The precipitated protein was

centrifuged and the protein pellet was dissolved in IN NaOH.

Finally suitable an-.cunt of NaOH dissolved protein was diluted to

24

1.0 ml with distilled water and 5.0 ml of alkaline copper

reagent was added. After 10 minutes, 0.5 ml of Folin-Ciocalteu

reagent was added. Absorbance was read at 750 nm after 20

minutes. Bovine serum albumin (BSA) was used as standard.

Uric Acid Estimation :-

Serum uric acid was estimated by the method of

Wootton (1964). Suitable amount of serum, 0.2 ml was diluted

upto 2.0 ml with distilled water and 0.325 ml of 0.66 N H2SO4

and 0.325 ml of 10% sodium tungstate were added. The contents

were kept for 10 minutes at room temperature and centrifuged at

3000 rpm for 15 minutes. Finally 1.0 ml of supernatant was

taken in another tube, to which 1.0 ml of 14% sodium carbonate

and 1.0 ml of uric acid reagent were added. Mixed well and

absorbance was taken after 15 minutes at 680 nm. For standard

plot, uric acid (0-40 ug) was diluted upto 1.0 ml with water, to

which 1.0 ml of 14% sodium carbonate and 1.0 ml of uric acid

reagent were added. Using this standard plot uric acid in serum

was calculated in terms of mg/100 ml serum. To prepare the uric

acid reagent, 10 gms of sodium tungstate and 1.0 gm of Na2HP0^

anhydrous was dissolved in 30 ml of distilled water. In a

second flask 2.5 ml of cone. H2SO4 was added to 10 ml water and

the diluted H2S0^ solution was added to the tungstate phosphate

solution and this mixture was reflexed for one hour. The

contents were diluted to 100 ml with distilled water.

Serum Albumin Estimation :-

Serum albumin was estimated by the method of Debro at al.,

(1557). 0.1 ml of serum was diluted to 2.0 ml with alcoholic -

TCA (1% TCA in 96% alcohol), mixed well by inversion and

centrifuged to sediment the precipitated globulin. Finally 0.1

ml of the supernatant was taken for Folin-Ciocalteu reagent (by

Lowry method). Albumin concentration was calculated from the

standard plot of bovine serum albumin.

Sulphahydryl Estimation :-

Total and free sulphahydryl (glutathione) were estimated

using the standard procedure described by Sedlak and Lindsay

(1968) .

(i) Total Sulphahydryl Estimation :- Liver homogenate in 0.02M

EDTA (0.5 ml of 2.5%) was mixed with 0.2M tris buffer (pH 8.2),

0.1 ml of dithio-2-nitrobenzoic acid (DTNB) (99 mg/25 ml

absolute methanol) and 7.9 ml of absolute methanol and kept at

room temperature for 15 minutes. After centrifugation at 3000

rpm for 15 minutes, absorbance of the supernatant was read at

412 nm.

(ii) Free Sulphahydryl Estimation :- To 1.0 ml of 2.5% liver

homogenate (in 0.02M EDTA) was added 1.0 ml of 10% TCA. The

test tubes were shaken intermittantly for 15 minutes at room

temperature and centrifuged at 3000 rpm for 15 minutes.

Supernatant (1.0 ml) was mixed with 1.4 ml of 0.4 M tris buffer

(pH 8.9). Finally 0.1 ml of DTNB was added and mixed well. The

absorbance was read at 412 nm.

Sulphahydryl content was calculated from the standard plot

of cysteine prepared in the same manner and expressed in terrs

of p, moles/gm liver.

26

cC-Tocopherol Estimation :-

oC-Tocopherol was measured both in liver homogenate and

serum by the method of Taylor et al., (1976). Liver homogenate

(1.5 ml of 10%) in 0.02 M EDTA was used in reaction mixutre.

Reaction mixture contained 1.0 ml of 23% ascorbic acid, 2.0 ml

of absolute ethanol, 1.5 ml of 10% of liver homogenate and 1.5

ml of tris-maleate buffer (50 mM, pH 6.5). Reaction mixture was

incubated at 70°C for 5 minutes. After that 2.0 ml of 10 N KOH

was added and incubated for 30 minutes at 70°C. Finally cC. -

tocopherol was extracted in 4.0 ml of n-hexane by mixing for 1

minute on a vortex mixer and then centrifuged for 5 minutes at

2000 rpm. Portion of hexane phase was removed for fluorometric

measurement at 286 nm excitation and 330 nm emission.

Before fluorometric measurement it is necessary to remove

the vitamin A interference. For this the hexane phase was

pipetted into the test tube and 0.6 ml of 60% H2SO. was added.

Samples were mixed vigorously for 30 seconds with a vortex mixer

and after centrifugation the fluorescence of hexane phase was

measured.

External standard was prepared by addition of 1.0 ml of

absolute ethanol that contained 2.5 ug oC-tocopherol to the 1.5

ml water and 0.5 ml of 25% ascorbic acid mixture. Tocopherol

concentration was calculated by comparing the sample

fluorescence intensity with that of the external standard after

correction had been made for the fluorescence intensity of the

blank. Tocopherol recoveries were estimated from internal

standard of 2.5 }xg tocopherol added to 1.5 ml of subcellular

suspension.

27

Similarly, oC-tocopherol was also estimated in serum. An

aliquot of 0.2 ml of serum was saponified with 1.0 ml of 10 N

KOH at 70°C for 3 0 minutes and finally oC -tocopherol was

extracted in 4.0 ml of n-hexane. .-.csorbance of hexane phase was

taken at 286 nm excitation and 330 nm emission.

Ascorbic Acid Estimation :-

Ascorbic acid in liver horr.cgenate was measured by the

method of Schaffert and George (1955). Liver homogenate (0.5 ml

of 10%) in 0.02 M EDTA was added in 4.5 ml of 6% TCA, mixed

well and kept for 5 minutes at room temperature. After that

tubes were centrifuged for 15 minutes at 3000 rpm. Supernatant

was collected in separate tube to which activated charcoal was

added and filtered. To 1.0 ml of filtrate in another tube, 3.0

ml of 4% TCA and one drop of thiourea were added. After

thorough mixing of the contents, 1.0 ml of 2,4-dinitrophenyl

hydrazine was added. The tubes were boiled for 10 minutes on

water bath and placed in crushed ice. Finally 5.0 ml of 85% of

H2S0^, was added and absorbance was read at 515 nm after 10

minutes. Sample blank was prepared in absence of filtrate. L-

ascorbic acid (0-60 p.g) in 4% TCA was used as standard.

Ceruloplasmin Assay :-

Ceruloplasmin was assayed by the method of Herbert and

Ravin (1961). Substrate, p-phenylenediamine-dihydrochloride

(PPD-2HC1) was dissolved in a minir.um volume of hot distilled

water, iacolorized with charccal, filtered while hot and

recrystallized from the water clear filtrate. The white

crystals of purified PFD-2HC1 were iried and stored in a vacuum

2 8

over caiciutii cl)ioride. A U.5% soiuLion oi tlie puiilied

substrate was prepared in distilled water just prior to use in

the test.

Serum (0.1 ml) was blown into the bottom of the test

tubes. Sodium azide (1.0 ml of 0.5%) was then added to one tube

to provide as enzyme inhibited control. Sodium acetate buffer,

8.0 ml (0.4 M, pH 5.5) was added to all the tubes, followed by

1.0 ml of 0.5% solution of substrate. The test tubes were

shaken to ensure thorough mixing and placed in water bath at

37°C. After 1 hour of incubation, 1.0 ml of 0.5% sodium azide

was added to each tube, excluding the control tube. The tubes

were shaken once more to ensure thorough mixing and were placed

in a refrigerator for 30 minutes at 4-10°C. The optical density

of the cooled reaction mixture was taken at 530 nm. Results

were expressed in terms of mg% ceruloplasmin in whole serum.

Ferroxidase Activity Assay :-

Ferroxidase activity in serum was assayed as described by

Gutteridge (1987) in the total reaction volume of 4 ml using 2%

rat brain homogenate in 0.15 M KCl as a source of phospholipid.

The reaction mixture for measuring ferroxidase activity

contained, 3.0 ml 2% rat brain homogenate (w/v) , 20 ;uM Fe' ' (as

ferrous sulphate) and 1.0 ml of serum. Total reaction mixture

was incubated for one hour at 37°C in a metabolic shaker.

Reaction was stopped by adding 1.0 ml of 20% TCA. Supernatant

was separated by centrifugation at 3000 rpm for 10 minutes. To

2.0 ml aliquot was added 1.0 ml of 0.67% (w/v) 2-thiobarbituric

acid (TBA) and TBA chromogen colour was developed by boiling the

29

tubes on water bath for 15 minutes. Optical density was

measured at 532 nm. The values are expressed as nmoles of

malonaldehyde equivalents generated by using molecular

absorption coefficient as 1.56 x 10 cm" M'-*-.

Catalase Assay :-

Catalase activity in liver homogenate was assayed by the

method of Luck (1974). Liver (1.0 gm) was homogenized in 18.0

ml of distilled water and to it 1.0 ml of 10% • deoxycholic acid

(DOC) was added. The homogenate was kept at room temperature

for 30 minutes. The homogenate was centrifuged at 35,000 rpm in

the fixed angle 50 rotor for one hour and the supernatant was

saved for catalase assay.

For catalase assay, in reference cuvette, 3.0 ml of H/15

phosphate buffer of pH 7.0 was taken and in the sample cuvette,

3.0 ml of hydrogen peroxide (H2O2) buffer was added. An aliquot

of 0.02 ml of supernatant of liver homogenate was added in

reference and sample cuvette and mixed well with the

micropipette and the time for the decrease in optical density

from 0.450 to 0.400 at 240 nm was quickly measured. Catalase

activity was calculated in terms of p, moles ^0^2 ^sduced per

minute per mg protein using extinction coefficient 0.036 mole~

cm'-'-L"- at 240 nm.

Estimation of Serum Iron-Binding Capacity :-

Serum iron binding capacity was estimated by the method of

Peters et al., (1956). To 0,2 ml of serum in the test tube 0.05

ml ferrous sulphate solution (equals 11.2 mg of iron/ml) was

30

added and allowed to stand for 15 minutes at room temperature.

After that 0.2 ml equivalent of the dry resin (Amberlite IRA 4]0

resin) was added and stirred occasionally for 5 minutes. Then

2.0 ml of 0.04M veronol buffer of pH 7,5 was added and continued

to stir for 5 minutes. The contents were centrifuged briefly

and 1.0 ml of the supernatant was taken using a manual pipette

control to avoid disturbing the resin. To 1,0 ml of above

supernatant, 1.0 ml of concentrated hydrochloric acid and 1.0 ml

of B-mercaptoethanol were added. The reaction mixture was kept

for 3 0 minutes at room temperature and precipitated with 1.0 ml

of 30% TCA. After 20 minutes the tubes were centrifuged at 3000

rpm for 10 minutes. After that 1,0 ml of supernatant was

removed and 0.45 ml of saturated sodium acetate was added.

Finally the colour was developed by adding 1.0 ml of

bathophenanthroline sulfonic acid solution (20 mg

bathophenanthroline sulfonic acid in 100 ml of isopropyl:

isoamyl alcohol) (3:1). Absorbance was taken after 10 minutes

at 535 nm. Blank tube contained 1.0 ml of distilled water, 0.45

ml saturated sodium acetate and 1.0 ml of bathophenanthroline

sulfonic acid solution. Standard iron tube contained, 0.5 ml of

ImM Fe solution, 0,45 ml of saturated sodium acetate, 1.0 ml

of bathophenanthroline sulfonic acid and diluted it upto 2.45 ml

with distilled water.

Estimation of Lipid Peroxidation :-

Lipid peroxidation was estimated in 10% liver homogenate

by the method described by Bernheim et al., (1948). Liver

homogenate (10%) was prepared in 0,15 M KCl, Liver homogenate

31

(10 ml) was taken in 25 ml conical flask and placed in metabolic

shaker water bath at 37°C. An aliquot of 1,0 ml of homogenate

was taken out at different time interval (i.e. 0, 30 and 60

minutes) and precipitated with 1.0 ml of 10% TCA. After waiting

for few minutes the tubes were centrifuged at 3000 rpm for 15

minutes. Finally 1.0 ml of aliquot was taken out and 1.0 ml of

0.6 7% (w/v) TBA was added. TBA chromogen colour was developed

by boiling the tubes on water bath for 15 minutes and absorbance

was read at 532 nm. Lipid peroxidation was expressed in terms

of nmoles/hr/gm liver using molar extinction coefficient

1.56x10^ cm'^M'^ at 532 nm.

Formation of Thiobarbituric Acid Reactive Product (TEAR) from Glutamate or Deoxyuridine :-

Formation of TEAR from glutamate or deoxyuridine in the

presence of 6 -aminolevulinic acid (ALA) with or without the

addition of copper ions was followed according to the method

described (Gutteridge, 1981; Quinlan and Gutteridge, 1987) . ALA

(0.2-1.0 mM) and copper ions (0.002-0.16 mM) were used as the

effective concentration to exhibit a linear progress of the

reaction for upto 2 hrs. Thus, all experiments noted in the

present studies were performed using 1.0 mM ALA, 0.16 mM copper

ions and 2 hrs. incubation except otherwise indicated. The

reaction mixture in a total volume of 1.2 ml contained 0.033 M

sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 4 mM glutamate

or 3.2 mM deoxyuridine, 1.0 mM ALA and oxyradical scavengers.

The reaction was initiated by the addition of CuSO^.5H2O to

provide a final concentration of 0.16 mM copper ion. The

contents were incubated for 2 hrs. at 37°C and the reaction was

32

terminated by the addition of 1.0 ml of 0.67% TBA (w/v) plus 0. "5

ml of 10% TCA (w/v). The contents were heated for L5 minuLea in

a boiling water bath and the resultant TBA chromogen was read at

532 nm using Spectronic 21 spectrophotometer. The values are

expressed as nmoles of malonaldehyde equivalents generated in 2

hrs. by using E532 = 1.56 x 10^ cm'-'-M"- .

Estimation of ALA :-

At the end of 2 hrs., incubation in the complete reaction

mixture ALA was estimated by the method of Mauzerall and Granick

(1956). The complete reaction mixture in a total volume of 1.2

ml contained 0.033 mM of sodium phosphate buffer in 0.15 M NaCl

(pH 7.4), 33 mM dimethyl sulfoxide and the reaction was

initiated by the addition of 0.16 mM copper ion. ALA was

estimated in 0.1 ml aliquot from the complete reaction mixture

by condensation with acetylacetone to form a pyrrole in a knorr

type reaction. Pyrrole was estimated by reaction with modified

Ehrlich's reagent and measurement of resultant colour at 555 nm

in Spectronic 21 spectrophotometer. A standard curve of ALA was

also run in the concentration range of 15-75 nmoles and the

values are expressed as nmoles of ALA utilized per 2 hrs.

Oxygen Uptake :-

Oxygen uptake in the complete reaction mixture was

followed with Clark electrode assembly on a Gilson Medical

Electronic Oxygraph. Percent oxygen consumption was estimated

in the reaction mixture containing 0.033 mM of phosphate buffer

in 0.15 M NaCl (pH 7.4), 4.0 mM glutamate and ALA (0.5 and 1.0

mM) . The reaction was initiated by the addition of 0.16 mM

33

copper ion. The means of at least two experiments are shown in

variation of 10% or less of the average.

Detection of Hydroxyl Radical :-

Hydroxyl radical ('OH) formation in the complete reaction

mixture during autooxidation of ALA was detected by the

formation of formaldehyde from dimethylsulfoxide (Klein et al. ,

1981). Incubation mixture of total volume (1.2 ml) contained

0.033 M sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 33 mM

dimethyl sulfoxide and the reaction was initiated by the

addition of 0.16 mM copper ion. After two hr of incubation

formaldehyde formed from dimethyl sulfoxide was estimated by the

method of Nash (1953). Formaldehyde formed was demonstrated by

the addition of 2.0 ml of Nash reagent (Ammonium acetate, 3 0

gms; acetylacetone, 0.4 gm; acetic acid 0.6 ml and water to make

up 100 ml) . After thorough mixing the contents were heated at

60°C for 30 minutes. The optical density was read at 415 nm.

The amount of formaldehyde formed was calculated from the

standard curve of formaldehyde which was run simultaneously.

Metal Analysis :-

Metals were analyzed in liver tissue of male and female

rats of benzene exposed and control group by the method

described by Herman (1980) . Equal weight of liver tissue was

added into the digestion flask containing 20 ml of digestion

mixture (1:6:: Perchloric acid (60^): nitric acid). The flasks

were boiled in the fuming chamber on hot plate till dry. The

dry ash was dissolved in 5.0 ml of 0.1 N nitric acid and

transfered into the test tube. The contents of iron and copper

34

were read using atomic absorption spectrophotometer. Results

were expressed in terms of p^g metal/gm liver.

S -ALA dehydratase Assay :-

6 -Aminolevulinic acid dehydratase activity was measured in

liver homogenate by the method of Gibson et al., (1955). The

enzyme activity was determined by measuring the amount of

porphobilinogen (PBG) formed after 1 hr at 37°C in 3.0 ml

reaction mixture containing 1.0 ml of 0.IM of. phosphate buffer

(pH 6.8), 10 ;amoles of 6 -ALA-HCl substrate neutralized with

buffer, 15 u moles of B-mercaptoethanol (0.33 ml/100 ml), 15 u

moles of KCl and 0.5 ml of 10% liver homogenate in 0.15 M KCl

(as enzyme source). Reaction was stopped by 1.0 ml of 20% TCA

containing 0.1 M mercuric chloride. In'control tube substrate

was added after adding TCA. To a measured aliquot, an equal

volume of freshly prepared Ehrlich's reagent was added and

optical density was measured at 555 nm using Spectronic 21

spectrophotometer. Results were calculated in- terms of nmoles

of PBG formed/hr/gm liver weight using molar extinction

coefficient 6.2 x 10 cm" M~ . To prepare Ehrlich's reagent 1

gm p-dimethylaminobenzaldehyde was dissolved in 30 ml of glacial

acetic acid and 16 ml of 70% perchloric acid and diluted it to

50 ml with glacial acetic acid.

Results

35

The level of antioxidant potentials of serum of rats

exposed to benzene by i.p. or s.c. are given in Table-1. Serum

uric acid level was found to be significantly decreased and the

maximum decrease of 52% was observed in the male group that

received benzene intraperitoneally. Serum albumin concentration

also ohowed decrease in male and female groups of rats exposed

to benzene. The extent of decrease is more significant in

female rats than in male rats.

The free sulphahydryl content and lipid peroxidation in

control and benzene exposed rats is given in Table-2. The free

sulphahydryl content of liver showed significant decrease

(P<0.05 in female; P <0.01 in male) in the groups which received

benzene intraperitoneally. However, the group which received

benzene s.c. did not show any significant decrease. Rate of

lipid peroxidation showed marked increase (2-4 fold) in all the

experimental groups when compared to respective control group of

animals.

36

c 0 H

n) U

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a) [/]

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£

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in

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38

Ceruloplasmin levels in serum, ascorbic acid level in

liver and catalase activity in liver (Table-3), and dC -

tocopherol levels in serum and liver (Table-4) did not show any-

significant change in any experimental group in comparison to

the respective control group of rats. 8 -Aminolevulinic acid

(ALA) dehydratase activity did not show any significant

alteration in liver of experimental group of male rats compared

tc the control group (Table - 5) . Earlier studies related to

alteration in iron metabolism and benzene exposure were done

only in female rats. In that context, ferroxidase activity and

iron binding capacity were studied in only female rats. Both

the groups that received benzene i.p. or s.c. showed that the

protection offered by serum was less against Fe catalysed

lipid peroxidation of brain homogenate than the serum from the

control group of animals (Table-6). Serum iron concentration

and total iron binding capacity was measured, which did not show

any significant change in benzene exposed animals with respect

to control group. Transferrin iron saturation in benzene

exposed rats showed values comparable with the control group.

Total iron and copper contents were estimated in liver

tissue of male and female rats exposed to benzene

intraperitoneally (Table-7). Only iron content showed

significant increase in male and female rats (P<0.02; P<0.05)

which received benzene i.p. and no significant change in copper

content was observed in either sex. Also iron and copper

contents were analyzed in isolated rat liver nuclei of benzene

exposed female rats (i.p.). In isolated rat liver nuclei only

iron content showed significant increase (P<0.05) but no change

39

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44

v\?as observed in copper content in isolated rat liver nuclei

compared to the respective control group.

TEAR formation from glutamate or deoxyuridine was observed

only in the presence of both 6 -aminolevulinic acid (ALA) and

copper ions which constitute the complete reaction mixture

(Table-8) . Absence of either ALA or copper ions did not show

any TEAR formation from glutamate or deoxyuridine. TEAR

formation from glutamate {Figure-2) was linear as a function of

time upto 2 h 30 min. and directly proportional to the

concentration of ALA (0.2-1.0 mM) though the TEAR formation from

deoxyuridine was more than two fold in comparison to glutamate

as a target molecule (Figure-3). Similarly TEAR formation from

glutamate increased linearly with the increase in concentration

of copper (2 0-160 uM) and that from deoxyuridine with 2 uM to 16

uM of copper (Figure-4 A and 4 B).

Oxygen consumption in the reaction mixture was not noticed

in the absence of copper. However, addition of copper resulted

in the consumption of oxygen and increase in oxygen consumption

v/as observed with the increase in ALA concentration (Figure 5) .

Generation of formaldehyde from dimethyl sulfoxide during

autooxidation of ALA was observed which was directly

proportional to the initial concentration and rate of

utilization of ALA (Table-9).

Inhibition of TEAR formation from glutamate or

deoxyuridine was observed in the presence of oxyradical

scavengers. Presence of thiourea, mannitol or catalase inhibited

TEAR formation from glutamate or deoxyuridine in the range of 22

to 99%. However, inhibition by benzoate, albumin or superoxide

45

00

1

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b w 0 0) -Q ^

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4J ^ '^ w rt

g 0 fn O 5

4J (U ™ ^ fl 0) 0 0 ^ 0 W

U) " • * U H

0 M -": t^ OJ rt

4-1 B X <U fl) n , 0) T)

S ^ H 0

iM -^ (1) 5 rd 0 g '

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46

120 150

Fig,

Time (min) Linearity of TBAR formation from glutamate as a function of time. The reaction mixture (total volume 1.2 ml) contained 0.033 mM of sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 4.0 mM glutamate and 1.0 mM ALA. The reaction was initiated by the addition of 0.16 mM copper ion. For details see Materials and Methods. The means of at least two experiments are shown with variation of 10% or less of the average.

47

A.0-

3.5

Q :

<

CD

10

o £

3.0-

2.5 J

2.0-

Fig. 3

0.2 O.A 0.6 0.8 1.0

ALA(mM) ALA concentration-dependent increase of TEAR formation from glutamate ( • • ) and deoxyuridine (0 0) . The reaction mixture (total volume 1.2 ml) contained 0.033 mM of soidum phosphate buffer in 0.15 M NaCl (pH 7.4), 4.0 nM glutamate or 3.2 mM deoxyuridine and 0.2-1.0 mM ALA. The reaction was initiated by the addition of 0.16 mM copper ion. For details see Materials and Methods. The means of at least two experiments are shown with variation of 10% or less of the average.

0.0^ 0.08 0.12 0.16

Cu^^CmM)

Fig. 4 A : Copper ion concentration-dependent increase of TEAR formation from glutamate. The reaction mixture (total volume 1.2 ml) contained 0.033 mM of sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 4.0 mM glutamate and 1.0 mM ALA. The reaction was initiated by the addition of 0.04-0.16 mM copper. For details see Materials and Methods. The means of at least two experiments are shown with variation of 10% or less of the average.

Cu2'*"( juM)

Fig. 4 B Copper ion increase of deoxyuridine. (total volume

concent rat ion-dependent TBAR fromation from The reaction mixture

1.2 ml) contained 0.033 mM of sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 3.2 mM deoxyuridine and 1.0 mM ALA. The reaction was initiated by the addition of 4-16 uM. For details see Materials and Methods. The means of at least two experiments are shown with variation of 10% or less of the averaoie.

50

100-

CD

>-. X

O

T ALA

80'

60-

/ . o -

20-

0 5 10

1^

15 20 25

Time (mm)

Fig. 5 : Oxygen consumption with respect to increase concentration of ALA. The reaction mixture containini? 0.033 mM of phosphate buffer in 0.15 U NaCl (pH 7.4), 4.0 mM glutamate and 0.5 mM ALA{A) or 1.0 mM ALA (B). The reaction was initiated by the addition of 0.16 mM copper ion. The means of at least two experiments are shown in variation of 10% or less of the average.

51

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0

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-H > O

03

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re

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0

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n H CN

O O

CN

O o

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Q 2

Ti (U 4J U

T i d • 0 <u U en

u d

u >

^ -d

Q^ X ^ (1) 0

M l t o

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So

UH 4-1 0

0) S CT,§

0) d 0

T3

j J 0 2

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p rH 0) rt iJ > ct!

u rH -H

< a

52

Table-10

Release of TEAR from glutamate or deoxyuridine during autooxidation of ALA in the presence of copper and the inhibition by oxygen radical scavengez"S.

Experimental conditions as described in Materials and Methods

nmoles TEAR formed after 2 hr incu­bation at 37°C^

Components

Complete reaction mixture

Urea (5 mM)*^

Thiourea

Mannitol

Benzoate

Albumin

Superoxide dismutase

Catalase

(5 mM)

(50 mM)

(50 mM)

(100 >ig/ml)

(100 Mg/ml)

(100 |ig/ml)

Glutamate

2.15

2.26

0.24

0.53

1.03

1.08

0.37

0.03

(89)^

(75)

(52)

(50)

(83)

(99)

Deoxyu:

4.04

3.93

0.15

1.56

3.84

3.63

3.98

3.16

ridine

(99)

(61)

(5)

(10)

(1)

(22)

^ All values are average of two sets of experiments conducted in duplicate with variations of 10% or less of the average.

Concentrations used are 0.033 mM sodium phosphate buffer in 0.15 M NaCl (pH 7.4), 4.0 mM glutamate or 3.2 mM deoxyuridine, 1.0 mM ALA and the reaction was initiated by the addition of 0.16 mM copper ion.

'^ Concentrations given in parentheses are those prior to the initiation of the reaction with copper.

Numbers in parentheses are percent inhibition.

53

dismutaae were s u b s t a n t i a l l y h ighe r (50 t o 83%) with

deoxyuridine. Urea was r e l a t i v e l y ine f fec t ive to i n h i b i t TEAR

fonnr^iion from both the ta rge t moleaules (Tab]e = 10).

Discussion

54

Benzene exposure has been shown to generate organic free

radicals and superoxide radical and it is generally accepted

that benzene requires metabolism to express its toxicity

(Subrahmanyan et al., 1991b). Polyphenolic metabolites of

benzene viz., hydroquinone and 1,2,4-benzenetriol have been

shown to be toxic and the suggested mechanism includes free

radical formation via superoxide during their autooxidation and

the covalent binding of semiquinones to DNA, RNA and other

cellular macromolecules (Wick, 1980; Rushmore et ai. , 1984). In

addition, the active oxygen radical formed during autooxidation

of polyphenolic metabolites has been shown to be involved in the

cytotoxic effects of benzene (Irons, 1985; Karam and Simic,

1989). Aerobic cells are equipped with an array of antioxidant

defences such as uric acid, albumin, cC -tocopherol, ascorbic

acid, free -SH groups, ceruloplasmin, catalase and ferroxidase

activity which terminate the propagation of damage by free

radicals (Halliwell and Gutteridge, 1990) .

55

Uric acid has been shown to be a selective antioxidant,

capable especially of reaction with hydroxyl radicals and

hypochlorous acid and also prevents oxidative inactivation of

endothelial enzymes, cyclooxygenase and angiotensin converting

enzyme (Becker, 1993). Uric acid can act as an antioxidant both

by binding iron and copper ions in forms that do not accelerate

free radical reactions, and by directly scavenging such as

singlet oxygen and peroxyl radicals (Ames et al., 1981). The

decrease in uric acid levels in serum of benzene exposed animals

may be due to scavenging action on oxyradicals generated during

benzene exposure. Howell and Wyngaarden (1960) showed that

methemoglobin or hematin in the presence of hydrogen peroxide

oxidized uric acid to allantoin, the initial step being a one-

electron oxidation of urate radical by a heme peroxide compound.

The consistent decrease in uric acid level in serum of benzene

exposed animals may be due to its rapid degradation by the free

radicals generated during the redox reactions of polyphenolic

metabolites of benzene. Low levels of uric acid may lead to

toxicity implications as physiological concentration of uric

acid has been shown to reduce oxo-heme oxidant, protect

erythrocytes from peroxidative damage leading to lysis and also

prevent oxidation of metmyoglobin by H2O2 (Arduini et al., 199

Serum albumin acts as an antioxidant through binding free

Cu "*" ions and site directs any radical damage onto albumin

molecule itself (Halliwell and Gutteridge, 1990) . Significant

decrease in serum albumin level of benzene treated rats may be

due to self degradation of oxidant-damaged albumin and may have

toxicological implications.

56

Protein sulphahydryl groups have also been suggested to

contribute significantly to the antioxidant capacity of plasma

(Wayner et al., 1987), although their oxidation could also be

considered as oxidative damage, depending on the protein

,iLJ (nM-ini. The cl<M.oxicjation of certain free radical met ab(jJ 1 ton

was shovm to be due to interaction of xenobiotic derived free

radicals with glutathione forming thiyl radical (GS*) (Ross,

1988; Wardman, 1988). The decrease in liver free -SH contents

in benzene treated rats is due to glutathione depletion through

oxidation of free radicals. cC -Tocopherol and ascorbic acid

are widely recognized as naturally occuring antioxidants in

biological systems. It has been hypothesized that oC-tocopherol

and ascorbic acid work synergistically to protect lipids from

peroxidation. oC -Tocopherol is an important lipid soluble

antioxidant and it breaks chains by trapping peroxyl and

alkyoxyl radical. The tocopherol radicals formed during the

biological processes are quenched and cC-tocopherols are

regenerated through interaction with ascorbic acid, this water

soluble ascorbate can act to spare depletion of membrane oC -

tocopherol levels ( Hartman and Shankel, 1990) . Moreover in

certain conditions the ascorbate radical can itself be

enzymatically reduced back to ascorbic acid by a NADH-dependent

system. There was no change in serum and liver oC-tocopherol and

liver ascorbic acid concentration in benzene exposed rats where

as several fold increase in rate of lipid peroxidation was

observed. This may be due to depletion of liver free -SH

contents in benzene exposed rats. The protective role of

catalase as an antioxidant also did not show any significant

57

change in liver of benzene exposed rats which catalyse the

conversion of hydrogen peroxide into water and oxygen.

Ceruloplasmin (EC 1.16.3.1) is an acute phase protein that

function as an antioxidant in biological system. The

antioxidant role of ceruloplasmin is thought to be due to its

forroxidase activity and has been shown to inhibit iron-

catalyzed lipid peroxidation, DNA damage and cellular protein

degradation (Gutteridge, 1991; Krsek-Staples and Webster, 1993).

The protection by ceruloplasmin during oxidative stress is

probably due to the oxidation of Fe II to Fe III and subsequent

prevention of the redox cycles necessary for hydroxyl or iron

radical formation; but no significant difference in

ceruloplasmin concentration in serum was observed in benzene

exposed rats compared to the respective controls.

Transferrin, an iron binding protein, transports iron in

the plasma. Iron bound to transferrin will not participate in

hydroxyl radical formation or lipid peroxidation (Aruoma and

Halliwell, 1987). In iron overload, serum iron concentration

increased with linear increase in transferrin saturated with

iron (Nielson et al., 1993). In benzene exposed rats (i.p. and

s.c.) no change in transferrin saturation, serum iron as well as

total iron binding capacity was observed, whereas significant

decrease in ferroxidase activity indicated decline in

antioxidant capacity of the serum to protect the radical

mediated peroxidation of biomolecules. The primary antioxidants

of normal plasma are concerned with limiting the reactivity of

iron (Gutteridge and Quinlan, 1993). ' i ^ ^ ' °" A.:; '' ^

58

Despite the large sex variations and route of exposure

observed in the biotransformation of xenobiotics by rats,

present studies revealed oxidant depletion in all the groups of

rats almost similarly (Sipes and Gandolfi, 1991) . The decrease

in antioxidant potentials in serum may allow oxygen

intermediates such as superoxide and hydrogen peroxide to

survive in plasma long enough. The significant decrease in uric

acid level in the present study may be of epidemiological

importance for clinical study in workers occupationally exposed

to benzene. Further studies are required to relate uric acid

levels in response to benzene exposure.

Trace amount of metal ions are present in biological

system. An estimated one third of all enzymes require a metal

ion in one or more phases for their catalytic activity.

Transition metal ions, most notably iron and copper can

facilitate the transfer of electrons to biological

macromolecules such as lipid, protein and DNA. In addition to

their biological function, metal ions are responsible for tissue

damage. Previous studies have suggested the involvement of iron

or copper in benzene metabolite-induced DNA damage through a

mechanism involving the generation of reactive oxygen species

(Kawanishi et al., 1989; Rao and Pandya, 1989). In the presence

oi trace metal ions the mutagenicity of polyphenolic metabolites

of benzene showed an increase to the tester strain Salmonella

tvphimurium (Lee and Lin, 1994). Recently Krsek-Staples and

Webster (1993) observed the iron-catalyzed oxygen resulting in

the formation of carbonyl derivatives with concomitant loss of

enzyme function.

59

It is suggested that the elevated level of low molecular

weight iron in the bone marrow during benzene toxicity may lead

to formation of tissue damaging species like lipid peroxide

radicals, superoxide anion (02~'), oxyferryl species, hydroxyl

radicals ('OH) which may form reactive metabolite(s) and may

react immediately if they occur in the vicinity of vital

molecules like nucleic acids (Lown and Sim, 1977) and polymerase

enzymes (Rushmore et al., 1984) which may be responsible for the

ultimate cytotoxicity. During in vitro study Rao and Pandya

(1989) obeorvod that polyphenolic metaboliteB of bonzenn a m

autooxidized faster in the presence of copper ions and resulting

in the formation of TEAR from target molecule such as glutamate

and DNA. The suggested mechanism includes free radical

formation via superoxide and the covalently binding of the

semiquinone metabolites of polyphenols to such molecules.

Significant increase in metal ions in liver and liver nuclei

during benzene exposure may have toxicological implications for

cytotoxicity of liver cells and liver nuclear DNA damage in

vivo.

In mammalian cells, g -aminolevulinic acid (ALA)

synthesized from glycine and succinyl CoA by ALA synthase in the

mitochondrial matrix, diffuses to the cytosol where it initiates

the pathway of porphyrin biosynthesis (Kappas et al., 1983).

Enhanced porphyrin synthesis and elevated urinary excretion of

ALA are usually associated with congenital erythropoietic

porphyria, intermittent acute porphyria and variegate porphyria

(Muller-Eberhard and Vincent, 1985). Biologically available

excess ALA, the first precursor of heme biosynthesis, has been

60

shown to cross the blood-brain barrier (McGillion et al. , 1974)

and accumulate in brain (McGillion et al., 1975). Xenobiotic

compounds including drugs and chemicals have been shown to alter

porphyrin metabolism with the accumulation of heme precursors.

ALA dehydratase, a cytosolic enzyme catalyzes the condensation

of two molecules of ALA to form the mono-porphobilinogen (PBG),

is inhibited by industrial pollutants such as lead (Monteiro et

al., 1991), benzene (Rao and Pandya, 1980),. trichloroethylene

(Fujita et al., 1984) and hexachlorobenzene (Elder, 1978) to

result in ALA accumulation and its loss in urine. It was

reported earlier that the decrease in hepatic ALA dehydratase

activity in female albino rats 3 h or 20 h after exposure to

benzene and suggested that an aromatic hydrocarbon epoxide may

react with the nucleophilic sulphahydryl groups of ALA

dehydratase (Rao and Pandya, 1980). Earlier Khan and Muzyka

(1973) reported increased levels of ALA, porphobilinogen and

protoporphyrin III in selected areas of rabbit brain following

chronic exposure to benzene and a significant rise of

erythrocyte ALA in industrial workers exposed to benzene. In

patients with intermittent acute porphyria and lead poisoning

where ALA accumulates, the erythrocytic activities of superoxide

dismutase and glutathione peroxidase are reported to be

increased and free radicals may be involved in the

symptomatology of neurological porphyrias and lead poisoning

(Monteiro et al., 1991). Bechara and Co-workers postulated that

ALA, being a source of reactive oxygen species (ROS) by metal

catalyzed aerobic oxidation in vitro (Monteiro et al., 1989) and

61

overproduced during acute intermittent porphyria (AlP) anc

hereditary tyrosinemia, (Hindmarsh, 1986; Fergusson, 1990). Ii

fact, ALA has been shown to induce lipid peroxidation and th«

release of encapsulated carboxyfluorescein from cardiolipin ricl

liposomes (Oteiza and Bechara, 1993), to induce single-stranc

breaks in plasmid pBR 322 DNA (Onuki at al., 1994), to caus(

iron catalyzed calcium dependent oxidative damage to the inne-

membrane of rat liver mitochondria (Hermes-Lima et al., 1992)

]t was observed that the plasma levels of ALA may attain uptc

100 fold higher in lead exposed ( >30 jig Pb/lOO gm blood]

individuals and symptomatic AIP patients than in norma]

individuals (Minder, 1986) and that, at least in the case oJ

rats, the liver concentrates ALA six times with respect tc

plasma (McGillion et al., 1975). Besides the plasma and liver,

ALA can also accumulate in heart, kidney, spleen, gut and fat

tissues (McGillion et al., 1975). In addition, ALA can cross

the blood- brain barrier and concentrates intraneuronally,

although its levels in cerebrospinal fluid are much more lowei

than those in plasma of porphyria patients in acute crisis. The

accumulation of ALA in nervous tissue has also been proposed anc

is probably responsible for the neurotoxicity observed ir

neurological porphyrias and lead poisoning. ALA has been showr

to undergo autooxidation at very high concentrations (6 mM) anc

this reaction is accelerated by the addition of oxyhemoglobir

and other iron complexes (Monteiro et al., 1989).

Copper ion concentration dependent oxygen uptake anc

disappearance of ALA during autooxidation of ALA (1.0 mM)

further corroborates that ALA autooxidation is associated witl:

6 2

the formation of active oxygen radicals. Earlier studies have

nhowp. that ALA uudorgoea enolization boforo conauming the

oxygen. A presemiquinoid nature of ALA has been reported

(Monteiro et al., 1989) because at physiological pH it undergoes

a Keto-enol tautomerism and subsequent autooxidation product has

the same reactivity as a semiquinone which can reduce oxygen to

superoxide anion or reduces cytochrome C via superoxide

(Monteiro et al., 1989).

Based on the present study it is proposed that the

interaction of copper ions with the amino group of ALA and

catalyse the oxidation of ALA. During this interaction cupric

ion forms a complex with ALA, which donates an electron reducing

it to the cuprous state. Molecular oxygen oxidizes the cuprous

ion to cupric ion with the formation of superoxide radical which

reduces metal ion as well as dismutates to form hydrogen

peroxide. Hydrogen peroxide could then interact with the

reduced copper to form a very reactive hydroxyl radical which

can attack glutamate or deoxyuridine to release TEAR products.

The release of TEAR products from glutamate or deoxyuridine by

ALA in the presence of copper ion suggests that ALA was acting

through the metal-catalyzed Haber-Weiss reaction in which

hydroxyl radicals are produced from hydrogen peroxide. The

presence of hydroxyl radicals in this system is also evident

from the formation of formaldehyde from dimethyl sulfoxide. The

two fold higher release of TEAR from deoxyuridine in comparison

to glutamate may be due to synergism among ALA, deoxyuridine and

oxygen free radicals as shown between bromodeoxyuridine and

paraquat (a superoxide-generating compound), to amplify free

63

radica] mediated UNA damage (Foot et al., 1989). This may

px-obably be the reason why oxyradical scavengers like benzoate,

albumin, superoxide dismutase and catalase were much less

effective in inhibiting deoxyuridine significantly as compared

to TEAR formation from glutamate.

Our observations indicate that ALA at physiological pH and

in the presence of copper can generate significant amounts of

reduced oxygen species. Cerebrospinal fluid taken from patients

v/ith neurological disorders contained micromolar concentrations

of loosely bound copper (Gutteridge, 1984) . Copper bound to

proteins in the biological milieu may also support this

autooxidation as it has been shown that copper containing enzyme

tyrosinase catalyse oxidation reaction (Wick and Fitzgerald,

1981). Kadiiska et al., (1993) and Kohen and Chevion (1985)

provided electron spin resonance spin-trapping evidence for the

synergistic generation of free radicals in vivo including the

hydroxyl radical, during copper-mediated paraquat toxicity.

Amplification of doxorubicin mutagenicity by cupric ion had

provided experimental evidence that anthracycline - metal ion-

DNA associations probably contribute to genotoxicity (Yourtee et

al., 1992). Intracellular concentrations of ALA capable of

releasing at least some of the iron (another predominant in vivo

transition metal capable of autooxidation and release of oxygen

radicals) embedded in the endoplasmic reticulum has been

suggested based on the observation that about 0.4 mM ALA can

release Fe(ll) fxom microsomes (Minotti, 1992). The present

observation may therefore help in improved understanding of the

64

p o s s i b l e mechanism of porphyr inpa thy and in v ivo t o x i c i t y

m a n i f e s t a t i o n of ALA, i f the a p p r o p r i a t e p resence and

in t e rac t ion of su i t ab le t r a n s i t i o n metal ion i s made ava i lab le

in tho liioloqical mil iau.

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Incli HI JoiiMi il of LxpirjiMciit il BioUi);) Vi.i t2 Much lyw pp m 2(m

Antioxidant potential in serum and liver of albino rats exposed to benzene

Sirfar.iz Ahmad, Vitiila Singh &G S Rao* IVlrokiiiii (Liitrgy Rciourtcs) Toxicity l/iduijlnal Toxicology Rcwarch Ccnirc PO Hox 80, Lutknow 226 (X)l, India

Rctcived 2 July 1993, revised 24 November 1993

Administration ofbcnzcnc (ip, 0 5 ml/kg body wt or sc, I ml/kg body wt) consecutively for 10 days to male and fLMi lie r its resulted in decrease in antioxidant potentials in serum Scnim line acid and albumin showed Mj;iiilii ml (ILLIL ISL m ill groups exposed tolKii/tne a-1 ocoplicrol levels did iiotcxliihit siniiilicantcli iiige in ui) ol tilt groups H lien Lompartd to control Intrcascin liver lipid peroxidation and decrease in content ol free sulphahydrjl group were observed in rats exposed to benzene Scrum ferroxidasc activity, total iron eoiiiL!il(l lC).ind tot.il iron binding cap icily (TIBC) in Tcmalc rats exposed to benzene showed signifitani iktre iseiii lerroxid iseaelivity wilhoutanyeli.mgcin IlCor I lUC wlieneoiiipareil toeontrol 1 lie decrease in .intioxid lilt poteiiti lis observed may be due to oxidation reaction exhibited by benzene metabolites, parli-eul 11 Iv liydriH|umone and I, 2, 4 ben/enetriol, resulting in oxidative stiCbs in ticiilcd iiits

Ol cupaiioii.ii exposure to bcii/cnc lias been associ.v-tcii with V,I nous biooclclyscrasia and.i variety of leu­kemia in hiirnan' ^ Bcn/cnc requires mclabolism to express ils (oxit efTccIs Ikn/cnc is primarily nictabol-i7ccl to phenol, liyclroqumonc, catechol and I, 2, 4-l)cn7eiiclfio! Recent sludics involve (lie polyplic-iiohc mclaluililcs as tlic loxic iiKcrmcdiatcs due to lliLii ca()il)ilily lo tiiidtfgo auto oxidation^ Hone iiiariowcoiilams several (leroxidascsand tlicpcroxi-d ilic mcl iholism of the benzene metabolites results Id llie /onit.ilioii ol seiiiic|uiii()iie Irtc r.idicals and oxygen atlivatioii to SII|KIOXRIC radicals'* Semiquin-oiic .iiid(| III none radicals lorincd during.luto-oxida-tioiiori'iclabolisinorp(>l)()licnolscan bind lo macro molecules such as protems, RNA and UNA' * For­mation ol scmiquinonc and ox)gen Irce radicals in bcn/cne exposed rats m ly lead to oxidative stress re­sulting 111 ilicred lion mct.ibolism which has been sliown dm mg benzene exposure' " Iron is released Il om iron stores under eoiidilions in which oxidative stress is involved'" Semicjuiiione radical of adriainy-cm has bee n sliown to iiiobiii/c and results in reduc­tive rcic isc ofiroii Irom Icrritin" Accumulation of low molecular weight iron and spontaneous genera­tion ofox)! KIK lis by bone marrow cells ofrats expo­sed to lien/eiie iMsbeeil re polled" I he low niolccu-

I ir weight iron is generally considered as decomparl-mentali/cd iron in contrast with iron bound lo pro­tein, ind IS citalytically active in miliating free radical II t e l l o i i s ilul l ipid pi l o x i d l l i o l l ' ' '*

* ( o i i L s p d i i i k i i l I I I I I K I I

Generation of free radical imposes depletion of anti­oxidants 7 he aim of the present study is to evaluate antioxidant potentials in serum and liver ofrats expo­sed to benzene

Materials and IVlctiiads All chemicals used were obtained cillicr from Sig­

ma Company, St Lotus, USA, or from (il.ixo Libor-atories, India (ANLAR)

SWISS VVistar strain albino rat,s ol either sex weigh­ing 100-110 g, bicd at the animal house unit of flic Centje(l I RC), wercdividcd mlo6gioups(3groups of male and 3 groups of female), of 6 animals each One group from each sex served as control One group from each sex received benzene (0 5 ml/kg body wt, ip)and another group (roni each sex received benzene (I ml/kg body wt.sc)daily for lOdays Alter the termi­nation of the treatment, blood w.is collected m non -hcp.irmed tubes from jugular vein and kept at 4°C Control and experimental rats weic sacrificed by cer­vical dislocation and liver and brain were immedia­tely excised and a 10% honiogcnatcwasmadeinO 15 M KCI to study lipid peroxid.ilion Liver liomoge-natc (10% w/v) was made in 0 02 M LDIA lor the estimation of free sulphahydryl content Brain ho-iiU)geiiatc(2yn w/v) was made 111 0 ISA/K( I as lipid source to study seiuiii lerroxidase activity

Liver and scrum a tocopherol were estimated" Serum uric acid'* and albumin''' were estimated us­ing the sl.indaid pi OK dims I eiioxid ise'" adivily ol ihr scf iiiii WHS UK isiir* d in Ihf lol il f( ,\( tiati vo lumcol4iiil which colli iincd 5mlol 2'/o biainhoiiio-

MM INDIAN J I X r i U O l MAI«( I I I'I'M

)H n III (\\ \ t ( M ' n i l o l l i I i i ' i i ' i i l p l i I d S d l i i l m i i (11

(•I VI I llll ll U M I U III! I l K l l l o l ' ( ) | l M I c ' ' .111(10 I m l (ll

M.I1I111 ,111(1 w . i x i m i i b i k i l 1(11 M) n u n . it "^TC l i i c

l I lKl l l II h l h l l K . K i d 11 K . l l \ ( p K u l l K l s \\(.'IC C s l l l l l -

.!((.(I I n I IK I I K I I U H I d l I k i i i l K i n i < / <;/ ' " 1 i \ c i l i i i i d

|KI(I \KI ilum' ' 111(1 liLCsulpli.iliy(li>r" wcicc^tmi.i-Icd iiiimcdi.ilcly Scrum iron binding cip.itily and idi ll null ut iccsliiii.iU'd iiMii)-' iMiluiphcniiiillitoline

nil i inu K id ''

RtMlllS

i^csulls .lie presented in lables 1 .ind 2 Scrum uriL .icid level signilicintly decreased and

the m.iximimi dctrc.ise ("i?"/,) w.is m m.ilcs which re­ceived bLii/cne ip(I.ible I) .Serum.illniminconccnt-i.ilion ,ils(i(ki.ic.i(.d III in.ik .iiid km.lie I.IISCX|H)SCII to Kn/ciK lliccxtciil ol (kcie.iscu.ismoicin Icinalc Ml' 111.in 111 m.ile r.il*.

i reesiilpli.ili\drvkoiitLiit ol liver showed signid-

(,iiil(ktKMsc(/ ' ' (Ml'iiiiliiiiilc, / ' - (MM III III,lie)III the jzMHi|)s wlnih a ie ivdl IKII/CIU' ip( I .iblc I) lldw ever, the jjioiips winch Kceived lK-ii/eiie s(. did mit show .my sif;nili(..iiil decit.ise K.ilcol lipid pctoxkl.i-lion showed m.iiketl iiRie.isc(2 4 lold) in .ill e\peii-menl.il gioiips when tomp.iied to respi,clive control group a-Tocophcrol levels in scrum and liver did not •ihow any chiiiifjc in iiiiy v\\w\ IIIUMIIIII (M oiip in i oni-|i.iiison loionl iol I'loiipol i.ils(mipnbli ,li(d d.il.l)

Larlicr studies related lo.illci.ilions iii iioii mcl.i-bolism and benzene cxposuie were dune only in lem-ale rats In that context, fcrroxidasc activity .\m\ iron bindmgcapacity were studied in only (cmalcrats The protection oHcrcd by scrum w.is less ng.iinsi I c^ c.il.i-lyscd lipid pcioxid.ilion ol biain liomogcn.ilc tli.in the sciiiin [loiii llic coiidol gioiip ol .iiiim.ils ( I ,iblc 2) .Scrum iron conccntr.ilion ,iiul lol.il iion binding t.ip.icity did not show .my sigmliL.iiit CII.IIIJ'L m benz­ene exposed .mini.lis with icsjicct to conliol group

I ihic I I (Id I ol bcii/<nc.ulii\inis(r ilKH! on .(.riiin concenlr.ition of .inlioxid.inC free - S H group .indst ile o( lipid pcroxid.ilioii

in Mis

jV ilins lit im in I SI v( (\ i iK I i( IIKS MI p iiiiillicsci .irc ptrtcnl iiitii ist ( + ) or (ILLK ISL ( ) over Lonlrolj

l',ir,inKl(r I cm lie M.ile

( oiilni)

Urn .icul (III) (II) M)') I 0 M ip SC

I'WMMK'' 2 4 4 1 0 i r ( - 1 6 ) ( - 2 1 )

Alininiiii (cAli) 4 87 + 0 19 4 11 + 0 14'' 3 88 + 0 15*' ( - M ) ( - 2 0 )

I K . SII minip ( " O i O I ' l 4 0(i±O24'' 4 6 6 ± 0 27'''' (|ini(ik/)Mi\ti) ( 11) ( - 0 1 )

I IJMI p, IOXMIIIIUII 2 11 Ml 1'. 1 11 | l I')'' 4 01 M>'1 '

|iiiiiM</hi/l I x d l I I I III) ( I M)

/' vi l lus II III ' II OS '•* Ni'l Mtiiilii ml

( (inlrol

5 0 7 1 0 18

3 9 1 + 0 15

3 54 + 0 16

2 P 10 11

2 4 5 1 0 18'

t - 5 2 )

3 51 + 0 09^

( - 1 1 )

' 2 5 4 1 0 19' ( -2K)

9 10 I I 5 1 '

( t mil)

1 1 1 1 0 12'

( 18)

1 7 2 + 0 20''' '

( - 0 5 )

144 + 0 22'''' ( - 0 ! )

5 01 i 1171"

( I I I'll

I il<l( '' Mlttl of bciuciK uliMimsu Uion on si rum fcrroxul isc .itlivily ind iron hindin)'cip icily m k m il( i iK

[V.ilucs arc m c . i n l S l ]

Ass.iy I crroxid.ise Iron binding c.ip.icily .iclivily*

(iiinoles MDA) Sci i i in I c

Oip/dl)

l l l k t (lig/dl)

I 111 iiiilu.Mu.jdi.ik H e ' ' 0 I iiiUtrum 17 l l 0 7X(n = 6) tonlrol 177111 ( n - 4 ) 9 S 2 1 125 (n 4) of control r.il'i

II |li.,ii, l i o n o K n i k N f ' ' Mi l ml 1 M | 0 SH'(n-^i) ip (•«|io«<'(l 16^ M I ( i i - f i ) KI9 | V ) ( „ - ( . ) <.c/iim ol ip < xiiosrd i.ih (16 7%)

III Br.iin homogcnalc l - t c ^ * + 0 I ml 42 4 +1 5'*'(n = 5) sc exposed 192 + 3 8 (n = 4) 9 0 3 ± 5 8 (n = 4) scrum of sc exposed rals (14 i%)

II ~ number o f r its

V.ilucs in pircnl)icsc<; arc percent incrcisc /•values "<0(H1|, *'<0 02 *l 'pi riiiii Ml ll (ll I Ilk ire pncn m M.ilcri.ik and Mclliods scclion t I ol ll null hiiiiliii|> ( ip II il> I I I III ill i n n l i on s i l i i i i i l ioi i

l i s t

t%)

18 6

211}

21 3

AHMAD <•/«/ ANIIOXIDANI I>01LNIIALINSLRUM&UVIR0I RAIS 2(15

11 iiiskrim iron s.itur.ilKMi m btn/ciic exposed rats sliowLtl \ lilies toinp ir.ibic wilh the control group

OlStllSSKIII Ikii/ciK (.\posuiL h.is Ixcii shown to generate or-

j ; line IK I I UIIL.IIS.IIKI superoxide raclit.il.iiui isgenc-lally aeecpled lli.il beii/eiie requires metabolism to i\pi(.ss lis io\ieil>' i'olvphenolic nielabolils of IKII /UU \\/ JisdioiiiiiMoiiL'and I, "* 4 ben/enelriol liavt Ixi iisliouii loix loKH and the suggested metlia-nisni iiH liidi s l uc radital loim ilion via superoxide diainf lliui auto oxid ilion and tlieeov.ileiit binding ai seinii[inn(Hic's (o DNA KNA aiul olliei Lclltil.ir 111 leioinoKeuies'- ' ' ' In addition the active oxygen r idieai lot incd during auto oxidation ofpolyphcno-lic metabohles have been sliou n to be involved m the eviolOKK UKttsol IK n/i iic ' ' '^ Aeiobie tells.iicc(|-uijipid Willi m a n ly ol aiilioxid ml deleikessutli as uiicaeid ilbumm a toeopherol, fiee - SH groups and/crroxidaseactivil) aliieh terminate the propa­gation ol damage by frtc ladicals"

IJrieaeid h is been shown to be a seleetive antioxi­dant, capable especially ol reaction with liydroxyl radicals .md h) pochlorous acid and also prevents ox-id.itivcmaeliv.ilion olcndolhclialcn/ymescycloox-ygcii.isc ,iiul .ingiolciisin converting cn/yme^' The dccic.isc in uiie .icid levels m sciiim o( bcn/cneexpo­se danim.ilsm.iy beduetosc.i\engmgactiononoxyr-.ulic.ils generated during bcn/cne exposure Low lev­els ol unc atid may lead to toxicitv implications as pliysiolojMt il coiKLiilr ilioiis ol uric .leid h.is been shown to iu(iieeo\i> heme oxid.ml piotecleiytliro-eyli gliosis Iroin lipid (leioxid.it on .indeivthrocytes fiom peioxidativc damage le idmg to lysis and ;ilso pievent oxidation ol nielmyoglobm by iliO^Cicf 28) Antioxid mt .leliviiy ol .ilbuinin is due to radical scav-cngmg.icdwty wlicrcin (hcictncoxsgcn radicalsdc-gr.ide .ilbuinm molecule itsclP'' Signilieant decrease m sciiim ilbiimin in bcn/enc lie ileil rats m.iy have loxicoloi'K il implii. ilioiis

I'lolem siilph ihvdisl gnuips h.ue.ilso been sugge-sled In (ooliihiile signilic.inlly to !hc .iiilioxid.mt t ip u il> 111 |)1 I m i ' ' ill hough I he II oxid ilion could .il obeeoii ideinl isoxid Uivcd.im.ige, depending on thcprolun .ilUcdd I hedotoxic it ion ofccrtam free r idical mc( iboliles was shown to be due to inlcr<ic-tion ol \cnobiotic derived fret radicals with glutathi­one lorminglhi)l radical (GS ) ' " • " The decrease in l iu r l r te S!l iToupiii h n/cnclici(cdralsisdiicIo glut ithioiiL (k plctioii tluoiii'h oxid.ilion oflrcc radi-c lis Ihcie w isnoch.mgc m serum and lis e ra locop-lurol in bt n/( lie cxp(isul i ils where is sever.il fold iiu u I SI 111 I ill (il lipid puoMil iiion w IS obsi I Mil lliism IV be due lodepleiioii ol li\c/ llcc Sll group m ben/ene ex|i()sctl i.ils

1 ranslcrrm, an iron binding protein, tr.insporl the iron in the pl.ism.i lion boiinil to li.iiislcrriii will not particip.ite in Ol I radic.il rorm.ilion or lipid peroxi-d.ilioii'^ In iron overlo.id, serum ironconcentr.ilion increased with line.ir mcrc.ise in tr.mslerrm s.iturated with i r o n " In ben7ene exfiosed i.ils (ip and sc) no ch.mge in Ir.insferrm s.iluralion, serum iron ,is well as tot.il iron biiulingc.ip.icily w.isobscived, where.is sig-mlic.mt(let(CISCIII Itnoxid.ise.iclivKy mdlc.l(edde tllnelll antioxKl.inl t.ip.icily ol the stiiim to piolect (he radical medi.iled petoxid.ilion ol biomoleciiles I liepnin.iiy .mtioxid.mlsol noim.il pi,ism i ,iie conc­

erned with limiting Ihe ic.ieliviiy ol iion' ' ' Despite the large sex variations and loute ol expos­

ure observed in the biotranslonnation of xenobiotics by Mis, present studies reve.ilcd .mlioxid.ml (Itplelioii in all Ihegioupsol inlsiilmosl siiiiil iily " I lu dti iiase 111 aiUioxid.ini potcnii.ils in sei iim m.iy .illovv oxygen intermediates such .11 superoxide .ind hydrogen per­oxide to survive in plasma long enough 1 he signilicanl decrease in uric .icid level in the present sliitly m.iy be ofepidemiologic.il import.nice in workers oceupa-(lonally exposed to ben/ene / urther studies arc re­quired to rel.ite uric acid levels m response to expos­ure to ben/ene

Acknowledgement 7 hanks arc due to fCMK, New/X'llii for grant iii-

did and Mr Lakshmi Kant for typographical assis­tance

Kcfcrcnces 1 RinskyRA Sniill iAl) lloriuinc K l o l l o i i K , Ymiiif'RJ,

Okun A II & I iiulier.m I'J /Vi ii /iif;l I MctI M6 (I^K?) 1044

2 Aksoy M liuinm lllili l\r\iHcl «2 (I9S9) 191 1 R lo G S A I'lnily,! K P l,ixuohR\ V) (198';) V)

4 Siibr.ihm iny im V V Rdss 1) I ISIMIDIUI I) A .^ Sniilli M I //<( «,/,/»,(/ Ihol M,,l l (IWI) 1'»S

5 Aircllinid dr i l l s ( ol icci A M i/yullo M <St I'lodi Ci (<iiHtr IJIIIIH (19KS) 159

r i j D w i l VViiikl( S K i l l ( , Wil / (p ,i& Siivacr It lliol ,H,il iiiHIiM mil mil i/iiid \ Vol III (I'll MIJlii I'll ss Niw Ynik) 1986 X25

7 [jcxl W K(K>:i'! J J 4% Siiyilir R,/(MKoZ/f/yi//Vi(jf»i(;(()/ 27 (1974)411

8 S n \ a s l a v i R C KlianS Sh inker U&Piiiily.i K P Anhloxt cnl 4 2 ( 1 9 7 9 ) 4 3

9 Pandya K P Rao G S Kh in S &. Krisliii inuirlliy R Arch loxuni 64(199(1) 119

10 I crrali M SignoiiniC ( l e t o l i l (^( oiiiiioiii M IhmlumJ 2X5(1992) 29S

11 Thorn is C t A A i i s l S n 'in h Bun In m Ihophy \ 21X(19«6) ( , t l

12 R l o d S ( IIHI S K iV I' iiuly I K P ( liti ( luin In \i>i ( iiiniti\ \ (I9;()) 111

II J.icohis A nio,H> 50 (1977)411

2( ) f ) I N D I A N J E X P B I O l . M A R C H I W 4

II II illiu>ll H.V (.Mil. 11.1)'. I M ( , \i.l,lluH}i,,n lUoj'hw ?'l(i ?0 ll.illiwi'jl II < (Inllcinlcc I M ( , in It IIUHIUII, lli„iiln\.2V. I r i ' (.1 III ( l i ) ' ) l l ) I

!•' I n l o i - I I 1111,1, , , M I'A l i | . | . , I M / , , . „ /> I I ( I ' l / d ) - ; ! ! ! 1? I l f i k n I I I h,i llinhial lliol M, ,1 |. | ( I ' / ' M ) f , | ' i

III \ \ i i n l l i ' i i M > I ' Mh i,',ufiU u\ mill, ,/i, itlhiiiihiiiiiMi i (( l i i i a ' - 7X A f i l i i i i i l A , M I I I K I IK HI < i. K i id i iK i < i I ,1 ). i i iui/ i( i VV. ( I I M l i ' . l r i r i

lu l l I..11.1.Mil I 'K.I \'IkVMknw'-V, hvr lUiiliial lliiil Mul,\\{W>7)-W>

W D . h i . i l l ' l . n v i i H.V K iMi i c i A /1 ,ih (liii Miil.M){\^^l) 29 W i i y i i r r I ) I ) M , l l i i i l i u i (1 W , I I I K O U I K I I , l l u i c l ny L K &

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