Differences in the biotransformation of hexachlorobenzene (HCB) in male and female rats

Ch~most,here, W~1.10, ~o.7, pp 779 - 793, 1 9 5 l Printed in Great Britain

DIFFERENCES

0045-6535/91/070779-07~02.00/O ©1981 Pergamon Press Ltd.

IN THE B I O T R A N S F O R M A T I O N IN MALE AND FEMALE

E.Richter,

Institut Medizinische

G.Renner,

and M.Wick

und Toxikologie,

der L u d w i g s - M a x i m i l i a n s - U n i v e r s i t ~ t

D-8000

MGnchen

(HCB)

RATS

J.Bayerl,

fGr P h a r m a k o l o g i e

Fakult~t

NuSbaumstr.26,

OF H E X A C H L O R O B E N Z E N E

2, Federal

Republic

MUnchen,

of Germany

ABSTRACT I. Male and female 770 mg HCB/kg spleen

Sprague-Dawley

body weight.

rats were given orally within one month

Livers and pooled

and heart were analysed

samples

of kidneys,

3, 24 and 52 days after

the last dose

for HCB and its m e t a b o l i t e s . 2. P e n t a c h l o r o p h e n o l

(PCP),

pentachlorothiophenol

2,3,5,6-tetrachlorophenol found besides 3. There

exists

HCB in all organs a significant

livers

of male and female

higher

content

Also

decreased

PCTP c o n c e n t r a t i o n s

No marked

difference rats,

decrease

2,3,4,6-

and

(PCB) were

investigated. in the metabolite

mainly attributed

of PCTP in females

4. HCB c o n c e n t r a t i o n s

(PCTP),

(TCP) and p e n t a c h l o r o b e n z e n e

pattern

in

to a significantly

than in males.

slower

decreased

in female slower

than in male rat livers.

in females

than in males.

at all was found during 7 weeks of elimination

in

females. 5. Differences explanation and

female

in the b i o t r a n s f o r m a t i o n for the different

of HCB could

porphyrinogenic

rats.

779

thus be an

activity

of HCB in male

780

INTRODUCTION

Hexachlorobenzene its m a j o r

(HCB) is a w i d e s p r e a d

toxic effects

r e s e m b l i n g p o r p h y r i a cutanea f r e q u e n t l y as chemical genesis

environmental

is to cause a chronic tarda in man.2'3

inducer in animal

of p o r p h y r i a cutanea

a c t i o n of HCB, however,

was found. 5,6

HCB has been used

to e l u c i d a t e

the patho-

In addition,

in the m e t a b o l i t e

of

in rat studies a marked sex

to male rats.

female rats after s u b c h r o n i c

A c c o r d i n g to Koss et al.4

of HCB is a p r e r e q u i s i t e

Female rats d e v e l o p e d

lower doses of HCB as c o m p a r e d

sex d i f f e r e n c e

is closely

tarda. The m e c h a n i s m of the p o r p h y r i n o g e n i c

is still not u n d e r s t o o d .

i n d u c t i o n of the porphyria.

of male and

Therefore

studies

it seems very likely that b i o t r a n s f o r m a t i o n

difference

pollutant. I One of

p o r p h y r i a which

porphyria

earlier and at

T h e r e f o r e we a n a l y s e d

HCB a d m i n i s t r a t i o n

livers

to look for a

pattern.

M A T E R I A L S AND M E T H O D S

Animals

Male and female S p r a g u e - D a w l e y F.R.G.)

of about

commercial

rats

(Versuchstierzucht

300 and 260 g body weight,

rat diet

(Altromin,

Lage,

Wiga,

respectively,

F.R.G.)

Sulzfeld,

were given a

and water ad libitum.

Materials

Hexachlorobenzene

and its m e t a b o l i t e s

2 , 3 , 5 , 6 - T C P were purified by g.l.c..

PCB,

PCP,

by r e c r y s t a l l i z a ~ i o n .

PCTP,

2,3,4,6- and

Purity of HCB was >99,7 %

Anisoles and t h i o a n i s o l e s were p r e p a r e d by m e t h y l a t i o n of the

c o r r e s p o n d i n g phenols and t h i o p h e n o l s with d i a z o m e t h a n e .

Methods

Each 40 male and female rats were given orally w i t h i n one month nine doses of 0.3 m m o l l k g

(85.6 mglkg)

of h e x a c h l o r o b e n z e n e

arachis oil in time intervals of 3 or 4 days. received

killed.

30 H C B - t r e a t e d

Body weight was r e c o r d e d weekly.

blood was c o l l e c t e d tocrit tubes, 7

hemoglobin.

24 and 52 days after the

At the end of the e x p e r i m e n t

for d e t e r m i n a t i o n of packed

Clay Adams,

Three days after the

rats of each sex and all control rats were

Five male and female rats were killed

last dose.

in

Ten rats of each sex

arachis oil only and served as control.

last dose

dissolved

Parsippany,

Liver, kidney,

cell volume

(Micro-hema-

USA) and c o n c e n t r a t i o n of

heart and spleen were dissected,

and frozen at -20 °C until analysis.

weighed,

781

D e t e r m i n a t i o n of h e x a c h l o r o b e n z e n e

Whole

and its m e t a b o l i t e s

livers and pooled samples of kidney,

spleen and heart were

t h o u r o u g h l y ground with sea sand in a mortar, 0.1M

HCI and subjected

n-hexane

as solvent

treated with 500 ml of

to a s t e a m - d i s t i l l a t i o n - e x t r a c t i o n

for three hours.8

of HCB and its major metabolites,

(SDE) with

Under these conditions

chlorinated

benzenes,

recoveries

phenols and thio-

phenols were almost complete. The hexane extract was dried over sodium sulfate,

concentrated

nitrogen

Excess d i a z o m e t h a n e was

and treated with d i a z o m e t h a n e

removed with nitrogen. concentrated

The hexane was

pilot

coupled with a Varian

and by capillary gas c h r o m a t o g r a p h y study the amounts of m e t h y l a t e d

T h e r e f o r e no d i f f e r e n t i a t i o n

g.l.c°-m.s.

1700 gas c h r o m a t o -

(Fraktovap,

Carlo Erba).

phenols and thiophenols

he×ane extract after the SDE p r o c e d u r e was estimated. a n i s o l e s and only minor amounts

In a

in the

No c h l o r i n a t e d

of p e n t a c h l o r o t h i o a n i s o l e

between

under

further cleaned up by shaking with

sulfuric acid and a n a l y s e d by g.l.c.-e.c.,

(Varian CH5 mass s p e c t r o m e t e r graph)

in hexane.

were

found.

free phenols and t h i o p h e n o l s and the

c o r r e s p o n d i n g anisoles and t h i o a n i s o l e s was made.

C a l c u l a t i o n s were

based on the free phenols and thiophenols.

Statistics

For c o m p a r i s o n of m e t a b o l i c multifactorial

analysis

4004/151.

Statistical

Student's

t test.

patterns

in livers of male and female rats a

of variance was c a l c u l a t e d with a Siemens

e v a l u a t i o n of sample means was made by a two tailed

RESULTS

One female rat died during the seventh week after the last dose of HCB. No s i g n i f i c a n t relative

d i f f e r e n c e s were seen in body weight gain and a b s o l u t e and

(to body weight)

weight was c o n s i d e r a b l y

w e i g h t s of kidney,

increased

heart and spleen.

Liver

three days after the last HCB dose from

7 . 1 8 ± 1 . 4 7 g (2.34±0.36 % of body weight)

to 13.02±2.15 g (4.34±0.73 %) in

male rats and from 5 . 7 8 ± 0 . 5 4 g (2.40±0.20 %) to 7.60±1.74 g (3.13±0.60 %) in female

rats.

Packed cell volumes were s i g n i f i c a n t l y

54.3±2.4 % to 5 2 . 1 ± 3 . 1 % in female rats. different

reduced

from

in male rats and from 47.6±1.8 % to 44.6±3.0 %

C o n c e n t r a t i o n of h e m o g l o b i n was not s i g n i f i c a n t l y

between treated and u n t r e a t e d groups.

782

Table

I.

Concentration of HCB and its metabolites

in the tissues of

male and female rats after oral administration of 0.3 mmol/kg HCB dissolved

in oil every 3 to 4 days.

are given as mean and standard deviation heart and spleen.

Results

(x±s x) in the case of

liver and as single value of pooled samples kidney,

of nine doses

in the case of

Values are expressed as mg/kg tissue

(wet weight)

day sex

n

HCB

PCB

PCP

PCTP

TCP

liver 3

24

52

m

30

192.2±70.2

0.048±0.017

3.16±1.70

0.234±0.087

0.017±0.012

f

30

147.0±72.5

0.033±0.014

2.12±1.66

0.356±0.123

0.041±0.051

m

5

37.2±5.4

0.011±0.008

0.34±0.10

0.166±0.060

0.007±0.007

f

5

72.4±13.4

0.007±0.001

0.76±0.46

0.268±0.059

0.012±0.009

m

5

30.7±12.4

0.011±0.004

0.52±0.32

0.116±0.043

0.010±0.006

f

4

58.4±10.6

0.009±0.002

0.47±0.25

0.373±0.078

0.011±0.010

kidney 3

m

30

127.4

0.045

5.792

0.242

0.089

f

30

111.4

0.012

3.690

0.098

0.075

24/52 m

l0

32.1

0.006

0.488

0.161

0.006

f

9

47.5

0.008

0.254

0.061

0.018

heart 3/24/52

m

40

62.9

0.004

0.690

0.036

0.075

f

39

80.5

0.005

0.640

0.018

0.010

0.591

0.098

0.007

spleen 3/24/52

m

40

f .

.

.

.

.

.

.

.

.

.

.

.

64.7

39 .

.

.

.

Significantly

0.006

67.1 .

.

.

.

.

.

.

.

0.011 .

.

.

different

.

.

.

.

.

.

.

.

.

.

.

0.244 .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

from the corresponding

0.038 .

.

.

.

.

.

.

.

.

.

.

.

.

.

0.009 .

.

.

.

.

value for male rats p<0.05

7~3

The results of the q u a n t i t a t i v e in liver,

kidney,

determination

of HCB and its m e t a b o l i t e s

heart and spleen are s u m m a r i z e d

in table

I. Three days

after the last dose livers of male rats c o n t a i n e d s i g n i f i c a n t l y more HCB, PCB and PCP, but s i g n i f i c a n t l y rat livers.

less PCTP and TCP as compared to female

After 24 and 52 days

female rat livers had s i g n i f i c a n t l y

higher HCB and PCTP c o n c e n t r a t i o n s a n a l y s i s of pooled samples not be s t a t i s t i c a l l y

evaluated.

PCTP in these organs were rats.

A multifactorial

analysis

24 days after the last dose

In contrast

pattern

concentrations

(table 3).

Table 2.

analysis

HCB and its m e t a b o l i t e s

of

in female compared to male confirmed

the s t a t i s t i c a l l y

in male and female livers

(table 2). A c o r r e l a t i o n analysis

and between HCB and PCTP

Because of

heart and spleen could

to liver,

of v a r i a n c e

showed a s i g n i f i c a n t

Multifactorial

for kidney,

in no case higher

significant different metabolic

all m e t a b o l i t e s

as the livers of male rats.

the results

3 and

for HCB and

c o r r e l a t i o n only between HCB and PCP

of v a r i a n c e PCB,

PCP,

for the c o n c e n t r a t i o n s

PCTP and TCP between

of

livers

of male and female rats.

Days after

Number and sex

Degrees of

of animals

freedom

last dose

3

30 m a l e / 3 0

female

T-value

Significance

5154

20.556

p < 0.01

24

5 male/5

female

5/4

28.715

p < 0.01

52

5 male/4

female

513

4.985

p > 0.05

The presence of p e n t a c h l o r o b e n z e n e

and the m e t h y l a t i o n

c h l o r o p h e n o l and p e n t a c ~ l o r o t h i o p h e n o l , chlorothioanisole confirmed

pentachloroanisole

and penta-

in hexane e x t r a c t s of organs was q u a l i t a t i v e l y

by g.l.c.-m.s.

g.l.c.-m.s.

products of penta-

The amount of t e t r a c h l o r o a n i s o l e was to low for

In c a p i l l a r y gas c h r o m a t o g r a m s

2,3,4,6- and 2 , 3 , 5 , 6 - t e t r a c h l o r o a n i s o l e ,

peaks of both isomers,

were

found.

784

Table

3.

Linear r e g r e s s i o n a n a l y s i s

between HCB and its m e t a b o l i t e s

PCP and PCTP in livers of male and female rats

3 days after

the last dose.

Metabolite

Sex

Intercept

Slope

Correlation

Significance

coefficient

PCP

male

- 0.217

0.0178

0.755

p < 0.001

female

- 0.071

0.0155

0.698

p < 0.001

PCTP

male

0.0779

0.000813

0.654

p <0.001

female

0.2413

0.000782

0.461

p <0.05

DISCUSSION

After rats were r e p e a t e d l y treated with h e x a c h l o r o b e n z e n e females c o n t a i n e d of males.

s i g n i f i c a n t l y more p e n t a c h l o r o t h i o p h e n o l

Our e x p e r i m e n t s

the livers of than the livers

did not allow a d e c i s i o n as to wether a higher

rate of p r o d u c t i o n or a lower rate of e l i m i n a t i o n was the cause difference.

Pentachlorothiophenol

derivatives

are known as HCB m e t a b o l i t e s .

the g l u t a t h i o n e metabolite.9 thiophenol,

c o n j u g a t e of HCB,

for this

and some other s u l f u r - c o n t a i n i n g They a p p a r e n t l y

originated

from

its primary s u l f u r - c o n t a i n i n g

Although various m e t a b o l i t e s

of HCB,

including pentachloro-

o b v i o u s l y did not lead to the same d i s t u r b a n c e

in the

p o r p h y r i n m e t a b o l i s m as HCB by itself~01 other still unknown reactive intermediates metabolites, philic

of HCB degradation, could be responsible.

respectively

r e a c t i o n p r o d u c t s of its

Such s u b s t a n c e s

should have electro-

sites which e v e n t u a l l y could react with

sulfhydryl

groups of the u r o p o r p h y r i n o g e n

the n u c l e o p h i l i c 4 decarboxylase. The d i s t u r b a n c e

of the p o r p h y r i n pathway by HCB is mainly a t t r i b u t e d

to a d e c r e a s e

in the

a c t i v i t y of this enzyme. 3 A higher c o n c e n t r a t i o n of p e n t a c h l o r o t h i o p h e n o l and t h e r e f o r e of possible livers of female susceptibility

reactive

intermediates

of HCB m e t a b o l i s m in

rats could p o s s i b l y be an e x p l a n a t i o n

of female rats to the p o r p h y r i n o g e n i c

for the higher

a c t i o n of HCB.

ACKNOWLEDGEMENTS

The a u t h o r s are g r a t e f u l

to the funds of C.Bohnewand,

T . G r a v e n h o r s t and K . L . W e i g a n d

for their support.

E.Hauber,

G. and

7a5

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