Biotransformation of the fungicide pentachloronitrobenzene by Tetrahymena thermophila

Chemosphere,Vol.ll,No.l,pp Printed in Great Britain

33-39,1982

0045-6535/82/010033-07503.00/0 © 1 9 8 2 Pergamon Press Ltd.

BIOTRANSFORMATION PENTACHLORONITROBENZENE Sharon E. Murphy,

OF THE FUNGICIDE

BY TETRAHYMENA

AnnaMarie

THERMOPHILA

Drotar and Ray Fall

D e p a r t m e n t of Chemistry and Cooperative Institute for Research in Environmental Sciences, Campus Box 215, University of Colorado, Boulder, Colorado 80309 Sum/rLary. The protozoan, Tetrah[mena thermophila, metabolizes pentachloronitrobenzene to several products, including nitrite, p e n t a c h l o r o t h i o a n i s o l e and pentachloroaniline. The latter two metabolites were identified by gas chromatographymass spectrometry. P e n t a c h l o r o t h i o a n i s o l e may be produced via a glutathioned e p e n d e n t pathway, and two key enzymes of this pathway, g l u t a t h i o n e transferase and thiol S-methyltransferase, have been detected in crude extracts of this microorganism. Pentachloronitrobenzene infectant.

Residues

agricultural associated zene

commonly

pentachloroaniline

(i), shellfish are:

pentachlorobenzene

found as impurities

(PCA)

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

The little

information

that exists on the microbial

thiomethylation

ity occurs

in many eukaryotic

and of PCNB

of PCNB,

for other examples

of microbial

and suggests

(GSH)

33

(i0).

In

in F. ox~-

transferase

activ-

of PCNB.

(12) , we set out to This report describes

of PCNB by the protozoan,

that this process

in-

aureo-

(ii), and the idea that thio-

by GSH transferase

metabolism

and thiomethylation

solani

PCTA was detected

that glutathione

microorganisms

of PCNB could be initiated

the d e n i t r i f i c a t i o n thermophila,

PCNB,

metabolism

and Rhizoctonia

of PCNB to produce

With our recent demonstration

methylation

and h e x a c h l o r o b e n -

available

reports of the reduction of PCNB to PCA by Streptomyces

(9) and the fungi Fusarium oxysporum

the latter report s_loorum.

in soil and

Major compounds

(PCTA) , metabolites

(6-8).

faciens

(3).

(PCB)

in commercially

and animals

only brief

and seed dis-

have been detected

(2) and peanut butter

(4,5)

cludes

look

is a widely used soil fungicide

of PCNB and related compounds

products

with PCNB residues

(HCB),

in plants

(PCNB)

is linked

Tetrahxmena

to a G S H - d e p e n d e n t

pathway.

34

EXPERIMENTAL

PCNB was o b t a i n e d p r e p a r a t i o n was thiophenol

from A l d r i c h Chemicals.

removed by p r e p a r a t i v e TLC.

(PCTP) was o b t a i n e d

The HCB c o n t a m i n a t i o n

A technical grade of p e n t a c h l o r o -

from pfaltz and Bauer,

acid and further p u r i f i e d by p r e p a r a t i v e TLC. ation of PCTP w i t h d i a z o m e t h a n e . tin and HCI.

recrystallized

in a c e t i c

PCTA was s y n t h e s i z e d by m e t h y l -

PCA was synthesized by r e d u c t i o n of PCNB w i t h

H e x a n e was p e s t i c i d e

grade.

The GSH c o n j u g a t e of PCNB was syn-

t h e s i z e d by the m e t h o d of L a m o u r e u x and Rusness chromatography

in this

(solvent system:

(5) and p u r i f i e d by liquid

50% 0.05 M p o t a s s i u m p h o s p h a t e buffer pH 7.0;

A l t e x ODS r e v e r s e phase column). Tetrah~mena ing 2%

t h e r m o p h i l a B 1868

was

grown at 30°C in c u l t u r e m e d i a c o n t a i n -

(w/v)

proteose peptone

(Difco)

cell d e n s i t y

of a p p r o x i m a t e l y

8x104 cells/ml

106 c e l l s / m l

for enzyme assays.

washed

in i0 m M Tris-Cl,

twice

with washed

at final c o n c e n t r a t i o n s A typical assay involved

tion v o l u m e

studies

and

and

Whole cell i n c u b a t i o n s were p e r f o r m e d

that of the o r i g i n a l culture.

to i n c u b a t i o n s centration).

(Ciba-Geigy) , to a

for b i o t r a n s f o r m a t i o n

Cells were h a r v e s t e d by c e n t r i f u g a t i o n pH 7.4.

cells s u s p e n d e d in 10 mM Tris-Cl,

approximately

greater

and 30 mg/L S e q u e s t r e n e

pH 7.4, at a cell c o n c e n t r a t i o n PCNB d i s s o l v e d

of 1 uM or I00 uM

in acetone was

added

(2% final a c e t o n e con-

incubation with shaking of a 2 mL reac-

in a 25 mL test tube at 25°C.

At the t e r m i n a t i o n of the r e a c t i o n

than 90% of the cells w e r e alive and swimming.

At the s p e c i f i e d

times,

the e n t i r e r e a c t i o n m i x t u r e was e x t r a c t e d twice with 2 mL volumes of hexane, cells w e r e ed.

first removed by c e n t r i f u g a t i o n and then the s u p e r n a t a n t was

The h e x a n e e x t r a c t s were a n a l y z e d

an e l e c t r o n SP2250

c a p t u r e detector.

The oven temperature was

300°C and the i n j e c t i o n port temperature

N i t r i t e was

180°C,

of the m e t a b o l i t e s was p e r f o r m e d by gas c h r o m a t o g r a p h y - m a s s

silica,

SE54

instrument.

s i l o x a n e deactivated.

The column

mass

spectrometry.

A cold trap i n j e c t i o n was used.

n i t r i t e release

ferase by p r o d u c t i o n of [ 3 H ] - P C T A (New E n g l a n d Nuclear;

(13)

A reaction

the s u p e r n a t a n t to 1 mL.

GSH t r a n s f e r a s e

from PCNB;

(ii) by

and thiol S - m e t h y l t r a n s -

from PCTP and S - a d e n o s y l - L - [ m e t h y l - 3 H ] 14).

(GC-MS)

50 m fused

of PCTA and PCA for

The cells were removed by c e n t r i f u g a t i o n ,

E n z y m e s w e r e assayed by p u b l i s h e d procedures:

Verification

spectrometry

1.2 L of h e x a n e and the extract c o n c e n t r a t e d

following GSH-dependent

methionine

(13).

(0.2 m m ID) was

m i x t u r e of 600 mL was used to o b t a i n s u f f i c i e n t q u a n t i t i e s

was e x t r a c t e d w i t h

3%

the d e t e c t o r

250°C.

assayed by the p r o c e d u r e of Snell and Snell

on a H e w l e t P a c k a r d M S 5 9 8 2 A

extract-

for m e t a b o l i t e s by gas c h r o m a t o g r a p h y with

The glass column was six feet long p a c k e d w i t h

on 100/120 Supelcoport.

temperature

or

35

RESULTS AND D I S C U S S I O N

Recent reports have d e s c r i b e d the appearance of PCTA in a s s o c i a t i o n with the use of the fungicide PCNB

(1-3).

The c o n v e r s i o n of PCNB to PCTA i n v o l v e s

p l a c e m e n t of a n i t r o - g r o u p w i t h a -SCH 3 moiety. transformation

We have considered

in the b i o s p h e r e could be m i c r o b i a l l y mediated,

examine a c r o s s - s e c t i o n of bacteria, ability to r e l e a s e nitrite ing is the protozoan,

that such a

and have begun to

fungi, protozoa and microalgae

from PCNB.

for the

The first p r o m i s i n g strain in this screen-

T. thermophila.

As shown in Figure

i, intact, washed T. thermophila cells incubated with

PCNB r e l e a s e d nitrite into the m e d i u m over an extended incubation period. this e x p e r i m e n t

re-

approximately

12% of the added PCNB was d e n i t r i f i e d

In

in 18 hr.

In other e x p e r i m e n t s with a lower PCNB c o n c e n t r a t i o n and/or higher cell to PCNB ratio c o n v e r s i o n s

of 75% were noted,

route of PCNB m e t a b o l i s m in T.

suggesting that d e n i t r i f i c a t i o n

is a m a j o r

thermophila.

10

8

~PCTA detected a=nitrite released "

O

E 4

. . o T e T o T

4

,

,

,

,

,

8 12 16 Reoction time (hrs)

Figure I. M e t a b o l i s m of PCNB by '~. thermophila. Cells were incubated with 100 uM PCNB for 1 to 18 hr. Cells were c e n t r i f u g e d and the reaction m e d i u m was either assayed directly for nitrite or e x t r a c t e d with hexane and the amount of PCTA present d e t e r m i n e d by gas c h r o m a t o g r a p h y .

36

To e x a m i n e

the organic p r o d u c t s

gas c h r o m a t o g r a p h y c u b a t e d w i t h PCNB.

Typical

lites are evident:

PCA

as shown in Figure

resulting

results are shown in Figure

(retention time 3.8 min)

2 (bottom).

2.

and P C T A

Two m a j o r metabo(retention time 5 min) ,

to a u t h e n t i c

standards,

spectra of the b i o l o g i c a l l y p r o d u c e d PCTA and PCA were

published

s p e c t r a l data

(8).

we used

p r o d u c e d by cells in-

The i d e n t i f i c a t i o n of these c o m p o u n d s was

a c c o m p l i s h e d by c h r o m a t o g r a p h i c c o m p a r i s o n The m a s s

from m e t a b o l i s m of PCNB,

to analyze hexane e x t r a c t a b l e m a t e r i a l s

and by GC-MS.

identical with

W h e n boiled cells were used in the incubation,

only h e x a n e e x t r a c t a b l e c o m p o u n d d e t e c t e d was u n r e a c t e d PCNB

the

(Fig. 2, top).

The p r o d u c t i o n and e x c r e t i o n of PCTA by T. t h e r m o p h i l a was shown to increase with

time of incubation,

cells

i).

(Fig.

but did not parallel

To further explore

c e l l u l a r P C T A the e x p e r i m e n t

PCNB was

the production of c e l l u l a r versus

in Figure

of PCNB and PCTA were d e t e r m i n e d

r e l e a s e of n i t r i t e by the

3 was performed.

extra-

In this case the levels

in the cells plus m e d i u m or the m e d i u m alone.

rapidly taken up from the m e d i u m and at the e a r l i e s t time p o i n t

associated with

the cells.

a p p e a r a n c e of PCNB efficiency).

(especially w h e n c o r r e c t e d

time p a r a l l e l e d

for d i f f e r e n c e s

less than 10% of the original

of the PCNB was

concentration.

80% was the dis-

in e x t r a c t i o n

The u n m e t a b o l i z e d PCNB left in the cells plus m e d i u m after

i n c u b a t i o n was

was e x c r e t e d

The p r o d u c t i o n of PCTA w i t h

8 hr of

Approximately

20%

c o n v e r t e d to PCTA in 8 hr, and less than a third of this PCTA

into the medium.

the PCNB metabolites.

P r o d u c t i o n of PCA r e p r e s e n t e d

less than

10% of

From these results it is clear that other p r o d u c t s of

PCNB m e t a b o l i s m are p r e s e n t in the cells and/or medium.

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

re-

m a i n to be identified. The m e c h a n i s m of i n t r o d u c t i o n of a -SCH 3 moiety into the PCNB m o l e c u l e of interest, xenobiotic (15,16).

is

since t h i o m e t h y l a t i o n has recently been r e c o g n i z e d as a class of

transformations

in the case of numerous drugs

and foreign c o m p o u n d s

Both n o n - e n z y m a t i c and e n z y m a t i c routes for this t r a n s f o r m a t i o n have

been p r o p o s e d

(see ref.

The p a t h w a y established,

16).

for t h i o m e t h y l a t i o n of PCNB in T. t h e r m o p h i l a

remains

but may be similar to the e n z y m a t i c p a t h w a y d e s c r i b e d

to be

in onion

(12),

as in the f o l l o w i n g scheme: PCNB + S - ( p e n t a c h l o r o p h e n y l ) g l u t a t h i o n e S-(pentachlorophenyl)cysteine

~ PCTP ~ P C T A

Two of the key enzymes of this pathway have been tracts of T. t h e r m o p h i l a S-methyltransferase, spectively.

(Table i).

catalyze

~

These enzymes,

identified

in crude ex-

GSH t r a n s f e r a s e

and thiol

the first and last steps of the scheme above,

GSH t r a n s f e r a s e was assayed by m e a s u r i n g

the nitrite r e l e a s e d

refrom

37

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I---

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38

PCNB,

and the reaction was shown to be dependent on the presence of GSH, PCNB

and enzyme

(Table i).

Table

i.

GSH transferase and thiol S-methyltransferase

activities

in T. thermophila a

Enzyme and Reaction

Product Formation b

Conditions

nmol per hr experiment

A.

experiment

2

GSH transferase Complete

(GSH, PCNB, enzyme)

minus GSH

12.6

15.9

<0.2

<0.2

minus PCNB

0

boiled enzyme

B.

1

0

<0.2

<0.2

Thiol S-Methyltransferase Complete

6.45

(SAM c, PCTP, DTT c, enzyme)

minus PCTP

0.58

minus DTT

0.77 0.12

minus enzyme minus PCTP,

11.4

-

plus PCP c

0.28

aReaction mixtures contained crude extract of T. thermo~hila (0.31.0 mg protein per assay) prepared as previously described (ii) except 10 mM Tris-Cl, pH 7.4, was used as the extraction buffer and centrifugation was carried out 48,000 x g for 20-30 min. bFor GSH transferase nitrite formation was measured; for thiol Smethyltransferase formation of toluene soluble [3H]PCTA was measured. CAbbreviations: SAM, S-adenosylmethionine; and PCP, pentachlorophenol.

HPLC analysis of the GSH conjugates GSH transferase preparation

DTT, dithiothreitol;

of PCNB produced with a highly purified

has revealed two products,

S-(pentachlorophenyl) glutathione,

tentatively

identified as

and S-(tetrachloronitrophenyl)glutathione.

The latter compound would be produced by GSH displacement of chloride rather than nitrite,

a result seen with GSH transferase

ity that S-(tetrachloronitrophenyl)glutathione mediate

in PCNB m e t a b o l i s m

from peanut

represents

(5).

The possibil-

an important inter-

in T. thermophila remains to be established.

39

The crude e x t r a c t was also a s s a y e d ing the f o r m a t i o n of toluene s o l u b l e

for thiol S - m e t h y l t r a n s f e r a s e ,

[3HI products

by follow-

from the reaction between

PCTP and S - a d e n o s y l - L - [ m e t h y l - 3 H ] m e t h i o n i n e

(14).

d e p e n d e n t on e n z y m e and PCTP was observed.

A reducing agent such as dithio-

threitol was also r e q u i r e d p r e s u m a b l y Pentachlorophenol

As seen in Table

1 a reaction

to maintain PCTP in a reduced state

did not serve as a substrate when substituted

for PCTP.

(12). The

r a d i o a c t i v e p r o d u c t of the e n z y m a t i c reaction with PCTP was identified by TLC as PCTA. While the c o m p l e t e likely

that in T.

e n z y m a t i c pathway

thermophila

remains to be established,

i n t r o d u c t i o n of a -SCH 3 group into PCNB involves

the sulfur atom of GSH and the methyl group of S - a d e n o s y l m e t h i o n i n e . possible contribution enzymatic pathway Finally, isms

it seems

of m e t h i o n i n e

The

as the source of the -SCH 3 group in a non-

is under active consideration.

considering

that GSH transferases are w i d e s p r e a d

(ii) it seems p o s s i b l e

that s u l f u r - c o n t a i n i n g m e t a b o l i t e s

in the b i o s p h e r e may be due in large part to microbial

in microorganof PCNB detected

action.

ACKNOWLEDGMENT We thank Dr. Robert B a r k l e y for running the GC-MS analyses. This research was s u p p o r t e d by grants from the U n i v e r s i t y of C o l o r a d o Council on Research and C r e a t i v e Work, and the N a t i o n a l Institutes of Health (ES 02639). REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.

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1981)