1134
Specialia
O n t h e b a s i s of t h e s e o b s e r v a t i o n s we s u g g e s t e d sb t h a t t h e F e ( I I ) - i n d u c e d i s o m e r i z a t i o n 1 - + 2 m i g h t serve as a m o d e l for t h e t r a n s f o r m a t i o n of t h e p r o s t a g l a n d i n e n d o p e r o x i d e s t o t h e P G E ' s in v i v o u n d e r t h e i n f l u e n c e of F e ( I I ) - b a s e d e n z y m e s y s t e m s as s h o w n in t h e first r o w of S c h e m e I I . T h e lower p a r t of t h e S c h e m e a d u m b r a t e s o u r s u g g e s t i o n for t h e b i o s y n t h e s i s of P G X a n d t h e t b r o m b o x a n e s , u s i n g as a m o d e l t h e 2 o t h e r r e a c t i o n s of e n d o p e r o x i d e s i n d u c e d b y t h e F e ( I I ) - F e ( I I I ) r e d o x s y s t e m w h i c h h a v e b e e n discussed in t h e p r e c e d i n g paragraphs. T h u s in t h e a n i o n r a d i c a l C, p r o d u c e d b y o n e - e l e c t r o n r e d u c t i o n of P G H 2, a n a l t e r n a t i v e t o o x i d a t i o n to P G E is a t t a c k b y t h e C-9 o x y r a d i c a l on t h e d o u b l e b o n d of t h e side c h a i n a t t a c h e d t o C-9 in a m a n n e r a n a l o g o u s t o t h e r e a c t i o n A - + B of S c h e m e I. T h i s is c o n s i s t e n t
EXPERIENTIA 33/9
w i t h t h e a l t e r a t i o n of d o u b l e b o n d g e o m e t r y (cis-+trans). Subsequent oxidation by the enzyme-based redox system w o u l d e x p l a i n t h e f o r m a t i o n of P G X . T h r o m b o x a n e b i o s y n t h e s i s r e q u i r e s c l e a v a g e of t h e 11, 12-bond of a p r e c u r s o r d e r i v e d f r o m P G H ~ 4, ~, 10. T h i s c a n b e r a t i o n a l i z e d b y i n v o k i n g o n e - e l e c t r o n r e d u c t i o n of P G H , to t h e isomeric a n i o n r a d i c a l D w h o s e f r a g m e n t a t i o n (arrows) and subsequent oxidation by the enzyme-based r e d o x s y s t e m is a n a l o g o u s t o t h e loss of a n i s o p r o p y ! g r o u p (and o x i d a t i o n of t h e latter) e x h i b i t e d b y d i h y d r o ascaridole o n t r e a t m e n t w i t h FeSO4 is. T h e c i r c u m s t a n c e t h a t r i n g c l e a v a g e leads t o a n allylic r a d i c a l m a y conc e i v a b l y a s s i s t t h e m o d e of f r a g m e n t a t i o n of D. T h e s u b s e q u e n t s t e p s l e a d i n g t o t h r o m b o x a n e A 2 a n d A a are self-explanatory.
0
~i ~ '".
.
.
R' : OH
.
R' .: 0~
: OH
Fe ("nT), H* or - H., Fe ('nT)
OH
OH PGE
C
PGHz
~
Fe (rr)
O'~-9
o...N~/~ ~
R
'
(~"
~
R
Fe ('nl"),H +
'
6--
OH
o•H
R
_ n . ~r(~T, OH
OH
D
I
OH PGX
Fragmentation
9
(o.
OH
OH
~./R
Fe ~
9
Fe (11r),H + or -- H ~Fe (m)
~ / R
"
..'~
"
> +
~
R'
OH O.H
OH
: ..~__./R
i ...~/R
OH
H
OH
thromboxane B2
OH thromboxone
A2
R e d u c t i v e d e c h l o r i n a t i o n of c h l o r o b i p h e n y l o l s by r a t s M. Th. M. T u l p 1, W. A. B r u g g e m a n a n d O. H u t z i n g e r
Laboratory o/Environmental Chemistry, University o/ Amsterdam, Nieuwe Achtergracht 166, Amsterdam (the Netherlands), 77 December 1976 Summary. D e c h l o r i n a t e d p r o d u c t s were i s o l a t e d f r o m t h e u r i n e of r a t s t h a t were a d m i n i s t e r e d c h l o r o b i p h e n y l o l s , t h e p r i m a r y h y d r o x y l a t e d m e t a b o l i t e s of P C B in m a m m a l s , T h e m e c h a n i s m of c h l o r i n e loss f r o m c h l o r o b i p h e n y l o l s is d i f f e r e n t f r o m t h e m e c h a n i s m of d e c h l o r i n a t i o n v i a a r e n e oxides w h e r e b y c o n c o m i t a n t h y d r o x y l a t i o n is a l w a y s observed. A l t h o u g h e x t e n s i v e m e t a b o l i c d e c h l o r i n a t i o n is k n o w n to o c c u r w i t h c o m p o u n d s in w h i c h c h l o r i n e is n o t b o u n d to a n a r o m a t i c c a r b o n * , t h i s r e a c t i o n is m u c h less i m p o r t a n t in a r o m a t i c c h l o r i n e c o m p o u n d s 3. T h e p o s s i b i l i t y of r e d u c t i v e d e c h l o r i n a t i o n of a n a r o m a t i c c h l o r i n e c o m p o u n d w a s f i r s t l y r e p o r t e d in 1973 * for h e x a c h l o r o b e n z e n e (HCB), a n d r e c e n t l y u n a m b i g u o u s l y d e m o n s t r a t e d : p e n t a c h l o r o b e n z e n e , t e t r a c h l o r o b e n z e n e a n d a n u m b e r of
p o l y c h l o r o p h e n o l s were f o u n d as m e t a b o l i t e s f r o m H C B in r a t s s, ~. T h i s r e d u c t i v e d e c h l o r i n a t i o n was s h o w n to b e c a t a l y s e d b y a n e n z y m e l o c a t e d in t h e m i c r o s o m a l f r a c t i o n of liver, lung, k i d n e y a n d i n t e s t i n e 5. R e d u c t i v e d e c h l o r i n a t i o n of p o l y c h l o r i n a t e d b i p h e n y l s (PCB) is k n o w n o n l y as a p h o t o c h e m i c a l p a t h w a y L All d o c u m e n t e d cases of m e t a b o l i c c h l o r i n e loss f r o m P C B i n v o l v e c o n c o m i t a n t h y d r o x y l a t i o n v i a a n a r e n e oxide
15. 9. 1977
Specialia
i n t e r m e d i a t e 3,s. D u r i n g a s t u d y on t h e m e t a b o l i s m of 4,4"-dichlorobiphenyl in r a t s 9, we r e c e n t l y d e t e c t e d 4'c h l o r o - 3 - b i p h e n y l o l as m i n o r m e t a b o l i t e . This c o m p o u n d w a s also f o u n d in t h e urine of r a t s a f t e r feeding 4,4'd i c h l o r o - 3 - b i p h e n y l o l , t h e m a j o r m e t a b o l i t e of 4,4'd i c h l o r o b i p h e n y l in t h i s animal. Such a m e t a b o l i c react i o n c a n n o t be e x p l a i n e d b y a n a r e n e oxide m e c h a n i s m b u t suggests direct metabolic dechlorination.
c' 1 2'
c'Ho ~
~
~1
~
H
OH r
1
o'.o
I
/k
HO'~~OH
HO~OH Ho~~OH
MOngOl~ HOHo'~~C iOH
I n t h i s s t u d y w i t h 4 s t r u c t u r a l l y r e l a t e d c o m p o u n d s (all h a v e a chlorine a t o m in t h e o r t h o p o s i t i o n to a h y d r o x y group), we s h o w t h a t s u c h d e e h l o r i n a t i o n r e a c t i o n s are apparently more common. Materials and methods. 4 , 4 ' - D i c h l o r o - 3 - b i p h e n y l o l , 4,4'd i c h l o r o - 3 , 3 ' - b i p h e n y l d i o l , 3-chloro-4-biphenylol a n d 2,6dichloropheltol of h i g h p u r i t y (99.9% b y GC-MS), were dissolved in p e a n u t oil (oleum arachidis) a n d a d m i n i s t e r e d orally t o m a l e W i s t a r r a t s as a single dose of 100 Ing/kg. The a n i m a l s were h o u s e d in i n d i v i d u a l m e t a b o l i c cages a n d s u p p l i e d w i t h w a t e r a n d food ad l i b i t u m d u r i n g t h e e x p e r i m e n t a l period of 7 d a y s . Faeces a n d u r i n e were collected s e p a r a t e l y in 4 N sulphuric acid t o p r e v e n t microbial m e t a b o l i s m a f t e r e x c r e t i o n . Isolation, purificat i o n a n d m e t h y l a t i o n of t h e m e t a b o l i t e s were p e r f o r m e d as d e s c r i b e d 9,1~ W e used a H e w l e t t - P a c k a r d 5982 A GC-MS s y s t e m o p e r a t i n g in t h e E l m o d e a t 70 eV, e q u i p p e d w i t h a n 0.4 • 180 c m all glass c o l u m n , p a c k e d w i t h 0.2% C a r b o w a x 20 M on C h r o m o s o r b W 100-120 mesh. S t a r t i n g m a t e r i a l a n d t h e m e t h y l e t h e r s of t h e m e t a b o l i t e s were s y n t h e s i z e d b y r e a c t i n g a n aniline w i t h a m y l n i t r i t e in t h e p r e s e n c e of a n excess of a r o m a t i c r e a c t a n t lo-13. Results and discussion. GC-MS i n v e s t i g a t i o n of t h e m e t h y l a t e d urine e x t r a c t s s h o w e d t h a t 4,4"-dichloro-3b i p h e n y l o l ( c o m p o u n d 1, see scheme), 4,4'-dichloro-3,3'b i p h e n y l d i o l ( c o m p o u n d 2), 3 - c h l o r o - 4 - b i p h e n y l o l (comp o u n d 3) a n d 2 , 6 - d i c h l o r o p h e n o l ( c o m p o u n d 4) were d e c h l o r i n a t e d t o f o r m 4 ' - c h l o r o - 3 - b i p h e n y l o l ( l a ) , 4c h l o r o - 3 , Y - b i p h e n y l d i o l (2a), 4 - b i p h e n y l o l (3a) a n d 2c h l o r o p h e n o l (4a) r e s p e c t i v e l y . Loss of chlorine in c o m p o u n d s I a to 4 a was e v i d e n t f r o m t h e c h a n g e in t h e specific i s o t o p e clusters of t h e m o l e c u l a r ions. The p o s i t i o n of t h e h y d r o x y g r o u p s in t h e s e c o m p o u n d s was clear f r o m t h e specific f r a g m e n t a t i o n p a t t e r n s of t h e i r m e t h y l e t h e r s ~, 10,13, a n d was f u r t h e r a s c e r t a i n e d b y s y n t h e s i s . I n all cases t h e d e c h l o r i n a t e d p r o d u c t s were m i n o r m e t a b o l i t e s (see t a b l e ) - a n d , e x c e p t for 2 a , could o n l y be d e t e c t e d in t h e urine. Since d i r e c t d e c h l o r i n a t i o n p r o d u c t s were n e v e r o b s e r v e d in s t u d i e s w i t h c h l o r o b i p h e n y l s 3, it s e e m s t h a t a h y d r o x y g r o u p in t h e a r o m a t i c n u c l e u s is n e c e s s a r y for t h e loss of a chlorine a t o m via r e d u c t i v e dechlorillation.
H~176 1
OH 4
OH
OH
O
9
2
4a
4b
4
3
5 Cumulative relative urinary excretion 7 days after an oral dose of 100 mg/kg
6 7
Compound
1 2 3 4
1135
Unchanged compound*
Hydroxylated metabolites
Deehlorinated metabolites
(%)
(%)
(%)
16 37 70 64
72 56 25 21
12 7 5 15
*Percentages were calculated from peak areas in the total ion chromatogram.
8 9 10 11 12 13
A fellowship to the first author by the Organisation for the Advancement of Pure Research (Stichting Z. W. 0., The Netherlands) is gratefully acknowledged. T. Ohsawa, J. R. Knox, S. Khalifa and J. E. Casida, J. agric. Fd Chem. 23, 98 (1975). G. Sundstr6m, O. Hutzinger and S. Safe, Chemosphere 5, 267 (1976). R . L . Metcalfe, I. P. Kapoor, P. Y. Lu, C. K. Schuth and P. Sherman, Envir. Health Persp. d, 35 (1973). H.M. Mehendale, M. Fields and H. B. Matthews, J. agric. Fd Chem. 23, 261 (1975). R. Engst, R. M. Macholtz and M. Kujawa, Bull. Envir. Contain. Toxic. /6, 248 (1976). L.O. Ruzo, M. J. Zabik aud R. D. Sehuetz, J. Am. chem. Soc. 96, 3809 (1974). S. Safe, D. Jones and O. Hutzinger, J. Chem. Perkin 1, 357 (1976). M. Th. M. Tulp, G. SundstrSm and O. Hutzinger, Chemosphere 5, 425 (1976). M. Th. M. Tulp, K. Olie and O. Hutzinger, Biomed. Mass Spectrom. in press. O. Hutzinger, S. Safe and V. Zitko, J. Ass. Offic. Anal. Chemists 57, 1061 (1974). O. Hutzinger, S. Safe and V. Zitko, in: The Chemistry of PCB's, p. 93. CRC Press, Cleveland, Ohio, USA 1974. B. Jansson and G. SundstrSm, Biomed. Mass Spectrom. 1, 386 (1974).
1136
Speeialia
I n all c o m p o u n d s s t u d i e d , t h e h y d r o x y g r o u p a n d t h e c h l o r i n e a t o m were in t h e o r t h o p o s i t i o n r e l a t i v e t o e a c h o t h e r a n d n o f u r t h e r r e l a t i o n s h i p (e. g. m e t a a n d p a r a ) was i n v e s t i g a t e d . All c o m p o u n d s also g a v e h y d r o x y l a t e d m e t a b o l i t e s f o r m e d b y e i t h e r d i r e c t h y d r o x y l a t i o n or hydroxylation via arene oxide intermediates. Compound 4 a was f u r t h e r d e c h l o r i n a t e d t o p h e n o l (4b). F r o m comp o u n d 3 3 m o r e m e t a b o l i t e s f o r m e d v i a r e d u c t i v e dec h l o r i n a t i o n ( c o m p o u n d s 3 e, 3 f a n d 3 g, see scheme) were isolated. B y s t u d y i n g t h e specific f r a g m e n t a t i o n pattern, the relative intensities and the retention times a n d b y c o m p a r i s o n w i t h d a t a of similar c o m p o u n d s f r o m earlier s t u d i e s g, x~ 13, t h e s t r u c t u r e s of t h e c o m p o u n d s
EXPERIENTIA 33/9
s h o w n i n t h e S c h e m e could b e d e t e r m i n e d . I n all cases f i n a l p r o o f was o b t a i n e d b y s y n t h e s i s . B e c a u s e t h e comp o u n d s 3 b , 3 c a n d 3 d are m a j o r m e t a b o l i t e s , w h i l s t 3 a , 3 e , 3 f a n d 3 g r e p r e s e n t o n l y a few p e r c e n t of t h e t o t a l a m o u n t of m e t a b o l i t e s formed, m o s t likely 3 e, 3 f a n d 3 g were f o r m e d v i a r e d u c t i v e d e c h l o r i n a t i o n of 3 b , 3 c a n d 3 d r e s p e c t i v e l y , since t h e l a t t e r all h a v e a h y d r o x y g r o u p o r t h o to a c h l o r i n e a t o m . T h e p r e s e n t d a t a h o w e v e r , do n o t rule o u t f o r m a t i o n of 3 e , 3 f a n d 3 g v i a t h e 3', 4'e p o x i d e of 3 a , similar t o 3', 4 ' - e p o x i d a t i o n of 3. T h e r e l a t i v e a m o u n t s of t h e u n c h a n g e d c o m p o u n d s , t h e i r h y d r o x y l a t e d a n d d e c h l o r i n a t e d m e t a b o l i t e s isolated f r o m t h e u r i n e are g i v e n in t h e t a b l e .
B i o s y n t h e s i s of i p h o s p h a t i d y l e t h a n o l a m i n e f r o m l C D P - e t h a n o l a m i n e b y t h e
Golgi
complex
! l i v e r in v i t r o 1
of~rat ....
P a t r i c i a L. C h a n g ~, J e n n i f e r M. Sturgess a n d M. A. Moscarello 3
Research Institute, Departments o/Pathology and Biochemistry, The Hospital/or Sick Children, Toronto (Ontario, Canada), 4 February 1977 Summary. A Golgi-rictl f r a c t i o n f r o m r a t liver h a s b e e n s h o w n to s y n t h e s i z e p h o s p h a t i d y l e t h a n o l a m i n e f r o m C D P e t h a n o l a m i n e in vitro. T h e i m p l i c a t i o n s of t h e e x i s t e n c e of s u c h a p a t h w a y for t h e m e m b r a n e flow h y p o t h e s i s are discussed. T h e b i o s y n t h e s i s of p h o s p h o l i p i d s h a s b e e n s h o w n t o o c c u r in e n d o p l a s m i c r e t i c u l u m 4-s a n d in m i t o c h o n d r i a 7, w h e r e a s t h e Golgi c o m p l e x h a s b e e n r e p o r t e d to lack s o m e of t h e e n z y m e s r e q u i r e d in s u c h b i o s y n t h e s i s 5, 0. H o w e v e r , t h i s r e p o r t p r e s e n t s e v i d e n c e t h a t t h e Golgi-rich f r a c t i o n s from rat liver can synthesize phosphatidylethanolamine f r o m C D P - e t h a n o l a m i n e in vitro, d e m o n s t r a t i n g t h a t a t 1000
=o "~ B00 8
R
d
~ ' - -
1,2-diacyl-sn-glycerol + CDP-ethanolamine* ethanolamine phosphotransferase phosphatidylethanolamine* + CMP When CDP-ethanolamine was radioactively labelled w i t h x*C in t h e e t h a n o l a m i n e m o i e t y , t h e r e c o v e r y of radioactive phosphatidylethanolamine measured the a c t i v i t y of e t h a n o l a m i n e p h o s p h o t r a n s f e r a s e , a n e n z y m e r e s p o n s i b l e for t h e f i n a l s t e p of p h o s p h a t i d y l e t h a n o l a m i n e de n o v o s y n t h e s i s a c c o r d i n g t o t h e K e n n e d y p a t h w a y . Materials and methods. All s u b c e l l u l a r f r a c t i o n s were p r e p a r e d f r o m t h e livers of m a l e W i s t a r r a t s w e i g h i n g 200-230 g. T h e r o u g h m i c r o s o m a l f r a c t i o n s were p r e p a r e d b y D a l l n e r ' s m e t h o d s a n d Golgi-rich f r a c t i o n s b y t h e m e t h o d of Sturgess, e t a l ) . T h e p l a s m a m e m b r a n e frac-
600
~-._= 400
l e a s t t h e t e r m i n a l e n z y m e is p r e s e n t . T h e r o u g h e n d o p l a s m i c r e t i c u l u m s h o w e d s i m i l a r s y n t h e t i c a c t i v i t y while t h e p l a s m a m e m b r a n e s h o w e d n o i n c o r p o r a t i o n of C D P ethanolamine. T h e r e a c t i o n u n d e r s t u d y w a s as follows:
ss S s
S
1
200
2 10 20 Time of incubation
30 rain
3 4
Recovery of radioactive phosphatidylethanolamine from CDPethanolamine vs time of incubation with rough microsomal, Golgirich and plasma membrane fractions of the rat liver. Rough microsomal (R, 232 I~g of protein), Golgi-rich (G, 139 v.g of protein) or plasma membrane (P, 117 p.g of protein) fractions were each incubated at 37 ~ in a medium containing CDP-[14C]-ethanolamine and diglyceride (see Materials and methods) in quadruplicate. At each time interval: 0, 10, 20 and 30 min, incubation was terminated, phospholipids were extracted from the total incubation mixture and separated by TLC. The spot corresponding to phosphatidylethanolamine (PE) was scraped and counted in a liquid scintillation system.
5 6 7 8 9
Acknowledgments. This study was supported by the Medical Research COuncil of Canada. Present address: Department of Paediatrics, McMaster Medical Center, Hamilton, Ontario, Canada. Reprint requests to be addressed to Dr M. A. Moscarello, Research Institute, Hospital for Sick Children, Toronto, M5G 1X8, Ontario, Canada. G . F . Wilgram and E. P. Kennedy, J. biol. Chem. 238, 2615 (1963). L . M . G . Van Golde, B. Fleischer and S. Fleischer, Biochim. biophys. Acta 249, 318 (1971). L.M.G. Van Golde, J. Raben, J. J. Batenburg, B. Fleischer, F. Zambrano and S. Fleischer, Biochim. biophys. Acta 360, 179 (1974). W.C. McMurray and R. M. C. Dawson, Biochem. J. 112, 91 (1969). G. Dallner, in: Methods in Enzymology. Ed. S. P. Colowick and N. O. Kaplan. Academic Press, New York 1974. J.M. Sturgess, E. Katona and M. A. Moscarello, J. Memb. Biol. 12, 367 (1973).
Specialia
O n t h e b a s i s of t h e s e o b s e r v a t i o n s we s u g g e s t e d sb t h a t t h e F e ( I I ) - i n d u c e d i s o m e r i z a t i o n 1 - + 2 m i g h t serve as a m o d e l for t h e t r a n s f o r m a t i o n of t h e p r o s t a g l a n d i n e n d o p e r o x i d e s t o t h e P G E ' s in v i v o u n d e r t h e i n f l u e n c e of F e ( I I ) - b a s e d e n z y m e s y s t e m s as s h o w n in t h e first r o w of S c h e m e I I . T h e lower p a r t of t h e S c h e m e a d u m b r a t e s o u r s u g g e s t i o n for t h e b i o s y n t h e s i s of P G X a n d t h e t b r o m b o x a n e s , u s i n g as a m o d e l t h e 2 o t h e r r e a c t i o n s of e n d o p e r o x i d e s i n d u c e d b y t h e F e ( I I ) - F e ( I I I ) r e d o x s y s t e m w h i c h h a v e b e e n discussed in t h e p r e c e d i n g paragraphs. T h u s in t h e a n i o n r a d i c a l C, p r o d u c e d b y o n e - e l e c t r o n r e d u c t i o n of P G H 2, a n a l t e r n a t i v e t o o x i d a t i o n to P G E is a t t a c k b y t h e C-9 o x y r a d i c a l on t h e d o u b l e b o n d of t h e side c h a i n a t t a c h e d t o C-9 in a m a n n e r a n a l o g o u s t o t h e r e a c t i o n A - + B of S c h e m e I. T h i s is c o n s i s t e n t
EXPERIENTIA 33/9
w i t h t h e a l t e r a t i o n of d o u b l e b o n d g e o m e t r y (cis-+trans). Subsequent oxidation by the enzyme-based redox system w o u l d e x p l a i n t h e f o r m a t i o n of P G X . T h r o m b o x a n e b i o s y n t h e s i s r e q u i r e s c l e a v a g e of t h e 11, 12-bond of a p r e c u r s o r d e r i v e d f r o m P G H ~ 4, ~, 10. T h i s c a n b e r a t i o n a l i z e d b y i n v o k i n g o n e - e l e c t r o n r e d u c t i o n of P G H , to t h e isomeric a n i o n r a d i c a l D w h o s e f r a g m e n t a t i o n (arrows) and subsequent oxidation by the enzyme-based r e d o x s y s t e m is a n a l o g o u s t o t h e loss of a n i s o p r o p y ! g r o u p (and o x i d a t i o n of t h e latter) e x h i b i t e d b y d i h y d r o ascaridole o n t r e a t m e n t w i t h FeSO4 is. T h e c i r c u m s t a n c e t h a t r i n g c l e a v a g e leads t o a n allylic r a d i c a l m a y conc e i v a b l y a s s i s t t h e m o d e of f r a g m e n t a t i o n of D. T h e s u b s e q u e n t s t e p s l e a d i n g t o t h r o m b o x a n e A 2 a n d A a are self-explanatory.
0
~i ~ '".
.
.
R' : OH
.
R' .: 0~
: OH
Fe ("nT), H* or - H., Fe ('nT)
OH
OH PGE
C
PGHz
~
Fe (rr)
O'~-9
o...N~/~ ~
R
'
(~"
~
R
Fe ('nl"),H +
'
6--
OH
o•H
R
_ n . ~r(~T, OH
OH
D
I
OH PGX
Fragmentation
9
(o.
OH
OH
~./R
Fe ~
9
Fe (11r),H + or -- H ~Fe (m)
~ / R
"
..'~
"
> +
~
R'
OH O.H
OH
: ..~__./R
i ...~/R
OH
H
OH
thromboxane B2
OH thromboxone
A2
R e d u c t i v e d e c h l o r i n a t i o n of c h l o r o b i p h e n y l o l s by r a t s M. Th. M. T u l p 1, W. A. B r u g g e m a n a n d O. H u t z i n g e r
Laboratory o/Environmental Chemistry, University o/ Amsterdam, Nieuwe Achtergracht 166, Amsterdam (the Netherlands), 77 December 1976 Summary. D e c h l o r i n a t e d p r o d u c t s were i s o l a t e d f r o m t h e u r i n e of r a t s t h a t were a d m i n i s t e r e d c h l o r o b i p h e n y l o l s , t h e p r i m a r y h y d r o x y l a t e d m e t a b o l i t e s of P C B in m a m m a l s , T h e m e c h a n i s m of c h l o r i n e loss f r o m c h l o r o b i p h e n y l o l s is d i f f e r e n t f r o m t h e m e c h a n i s m of d e c h l o r i n a t i o n v i a a r e n e oxides w h e r e b y c o n c o m i t a n t h y d r o x y l a t i o n is a l w a y s observed. A l t h o u g h e x t e n s i v e m e t a b o l i c d e c h l o r i n a t i o n is k n o w n to o c c u r w i t h c o m p o u n d s in w h i c h c h l o r i n e is n o t b o u n d to a n a r o m a t i c c a r b o n * , t h i s r e a c t i o n is m u c h less i m p o r t a n t in a r o m a t i c c h l o r i n e c o m p o u n d s 3. T h e p o s s i b i l i t y of r e d u c t i v e d e c h l o r i n a t i o n of a n a r o m a t i c c h l o r i n e c o m p o u n d w a s f i r s t l y r e p o r t e d in 1973 * for h e x a c h l o r o b e n z e n e (HCB), a n d r e c e n t l y u n a m b i g u o u s l y d e m o n s t r a t e d : p e n t a c h l o r o b e n z e n e , t e t r a c h l o r o b e n z e n e a n d a n u m b e r of
p o l y c h l o r o p h e n o l s were f o u n d as m e t a b o l i t e s f r o m H C B in r a t s s, ~. T h i s r e d u c t i v e d e c h l o r i n a t i o n was s h o w n to b e c a t a l y s e d b y a n e n z y m e l o c a t e d in t h e m i c r o s o m a l f r a c t i o n of liver, lung, k i d n e y a n d i n t e s t i n e 5. R e d u c t i v e d e c h l o r i n a t i o n of p o l y c h l o r i n a t e d b i p h e n y l s (PCB) is k n o w n o n l y as a p h o t o c h e m i c a l p a t h w a y L All d o c u m e n t e d cases of m e t a b o l i c c h l o r i n e loss f r o m P C B i n v o l v e c o n c o m i t a n t h y d r o x y l a t i o n v i a a n a r e n e oxide
15. 9. 1977
Specialia
i n t e r m e d i a t e 3,s. D u r i n g a s t u d y on t h e m e t a b o l i s m of 4,4"-dichlorobiphenyl in r a t s 9, we r e c e n t l y d e t e c t e d 4'c h l o r o - 3 - b i p h e n y l o l as m i n o r m e t a b o l i t e . This c o m p o u n d w a s also f o u n d in t h e urine of r a t s a f t e r feeding 4,4'd i c h l o r o - 3 - b i p h e n y l o l , t h e m a j o r m e t a b o l i t e of 4,4'd i c h l o r o b i p h e n y l in t h i s animal. Such a m e t a b o l i c react i o n c a n n o t be e x p l a i n e d b y a n a r e n e oxide m e c h a n i s m b u t suggests direct metabolic dechlorination.
c' 1 2'
c'Ho ~
~
~1
~
H
OH r
1
o'.o
I
/k
HO'~~OH
HO~OH Ho~~OH
MOngOl~ HOHo'~~C iOH
I n t h i s s t u d y w i t h 4 s t r u c t u r a l l y r e l a t e d c o m p o u n d s (all h a v e a chlorine a t o m in t h e o r t h o p o s i t i o n to a h y d r o x y group), we s h o w t h a t s u c h d e e h l o r i n a t i o n r e a c t i o n s are apparently more common. Materials and methods. 4 , 4 ' - D i c h l o r o - 3 - b i p h e n y l o l , 4,4'd i c h l o r o - 3 , 3 ' - b i p h e n y l d i o l , 3-chloro-4-biphenylol a n d 2,6dichloropheltol of h i g h p u r i t y (99.9% b y GC-MS), were dissolved in p e a n u t oil (oleum arachidis) a n d a d m i n i s t e r e d orally t o m a l e W i s t a r r a t s as a single dose of 100 Ing/kg. The a n i m a l s were h o u s e d in i n d i v i d u a l m e t a b o l i c cages a n d s u p p l i e d w i t h w a t e r a n d food ad l i b i t u m d u r i n g t h e e x p e r i m e n t a l period of 7 d a y s . Faeces a n d u r i n e were collected s e p a r a t e l y in 4 N sulphuric acid t o p r e v e n t microbial m e t a b o l i s m a f t e r e x c r e t i o n . Isolation, purificat i o n a n d m e t h y l a t i o n of t h e m e t a b o l i t e s were p e r f o r m e d as d e s c r i b e d 9,1~ W e used a H e w l e t t - P a c k a r d 5982 A GC-MS s y s t e m o p e r a t i n g in t h e E l m o d e a t 70 eV, e q u i p p e d w i t h a n 0.4 • 180 c m all glass c o l u m n , p a c k e d w i t h 0.2% C a r b o w a x 20 M on C h r o m o s o r b W 100-120 mesh. S t a r t i n g m a t e r i a l a n d t h e m e t h y l e t h e r s of t h e m e t a b o l i t e s were s y n t h e s i z e d b y r e a c t i n g a n aniline w i t h a m y l n i t r i t e in t h e p r e s e n c e of a n excess of a r o m a t i c r e a c t a n t lo-13. Results and discussion. GC-MS i n v e s t i g a t i o n of t h e m e t h y l a t e d urine e x t r a c t s s h o w e d t h a t 4,4"-dichloro-3b i p h e n y l o l ( c o m p o u n d 1, see scheme), 4,4'-dichloro-3,3'b i p h e n y l d i o l ( c o m p o u n d 2), 3 - c h l o r o - 4 - b i p h e n y l o l (comp o u n d 3) a n d 2 , 6 - d i c h l o r o p h e n o l ( c o m p o u n d 4) were d e c h l o r i n a t e d t o f o r m 4 ' - c h l o r o - 3 - b i p h e n y l o l ( l a ) , 4c h l o r o - 3 , Y - b i p h e n y l d i o l (2a), 4 - b i p h e n y l o l (3a) a n d 2c h l o r o p h e n o l (4a) r e s p e c t i v e l y . Loss of chlorine in c o m p o u n d s I a to 4 a was e v i d e n t f r o m t h e c h a n g e in t h e specific i s o t o p e clusters of t h e m o l e c u l a r ions. The p o s i t i o n of t h e h y d r o x y g r o u p s in t h e s e c o m p o u n d s was clear f r o m t h e specific f r a g m e n t a t i o n p a t t e r n s of t h e i r m e t h y l e t h e r s ~, 10,13, a n d was f u r t h e r a s c e r t a i n e d b y s y n t h e s i s . I n all cases t h e d e c h l o r i n a t e d p r o d u c t s were m i n o r m e t a b o l i t e s (see t a b l e ) - a n d , e x c e p t for 2 a , could o n l y be d e t e c t e d in t h e urine. Since d i r e c t d e c h l o r i n a t i o n p r o d u c t s were n e v e r o b s e r v e d in s t u d i e s w i t h c h l o r o b i p h e n y l s 3, it s e e m s t h a t a h y d r o x y g r o u p in t h e a r o m a t i c n u c l e u s is n e c e s s a r y for t h e loss of a chlorine a t o m via r e d u c t i v e dechlorillation.
H~176 1
OH 4
OH
OH
O
9
2
4a
4b
4
3
5 Cumulative relative urinary excretion 7 days after an oral dose of 100 mg/kg
6 7
Compound
1 2 3 4
1135
Unchanged compound*
Hydroxylated metabolites
Deehlorinated metabolites
(%)
(%)
(%)
16 37 70 64
72 56 25 21
12 7 5 15
*Percentages were calculated from peak areas in the total ion chromatogram.
8 9 10 11 12 13
A fellowship to the first author by the Organisation for the Advancement of Pure Research (Stichting Z. W. 0., The Netherlands) is gratefully acknowledged. T. Ohsawa, J. R. Knox, S. Khalifa and J. E. Casida, J. agric. Fd Chem. 23, 98 (1975). G. Sundstr6m, O. Hutzinger and S. Safe, Chemosphere 5, 267 (1976). R . L . Metcalfe, I. P. Kapoor, P. Y. Lu, C. K. Schuth and P. Sherman, Envir. Health Persp. d, 35 (1973). H.M. Mehendale, M. Fields and H. B. Matthews, J. agric. Fd Chem. 23, 261 (1975). R. Engst, R. M. Macholtz and M. Kujawa, Bull. Envir. Contain. Toxic. /6, 248 (1976). L.O. Ruzo, M. J. Zabik aud R. D. Sehuetz, J. Am. chem. Soc. 96, 3809 (1974). S. Safe, D. Jones and O. Hutzinger, J. Chem. Perkin 1, 357 (1976). M. Th. M. Tulp, G. SundstrSm and O. Hutzinger, Chemosphere 5, 425 (1976). M. Th. M. Tulp, K. Olie and O. Hutzinger, Biomed. Mass Spectrom. in press. O. Hutzinger, S. Safe and V. Zitko, J. Ass. Offic. Anal. Chemists 57, 1061 (1974). O. Hutzinger, S. Safe and V. Zitko, in: The Chemistry of PCB's, p. 93. CRC Press, Cleveland, Ohio, USA 1974. B. Jansson and G. SundstrSm, Biomed. Mass Spectrom. 1, 386 (1974).
1136
Speeialia
I n all c o m p o u n d s s t u d i e d , t h e h y d r o x y g r o u p a n d t h e c h l o r i n e a t o m were in t h e o r t h o p o s i t i o n r e l a t i v e t o e a c h o t h e r a n d n o f u r t h e r r e l a t i o n s h i p (e. g. m e t a a n d p a r a ) was i n v e s t i g a t e d . All c o m p o u n d s also g a v e h y d r o x y l a t e d m e t a b o l i t e s f o r m e d b y e i t h e r d i r e c t h y d r o x y l a t i o n or hydroxylation via arene oxide intermediates. Compound 4 a was f u r t h e r d e c h l o r i n a t e d t o p h e n o l (4b). F r o m comp o u n d 3 3 m o r e m e t a b o l i t e s f o r m e d v i a r e d u c t i v e dec h l o r i n a t i o n ( c o m p o u n d s 3 e, 3 f a n d 3 g, see scheme) were isolated. B y s t u d y i n g t h e specific f r a g m e n t a t i o n pattern, the relative intensities and the retention times a n d b y c o m p a r i s o n w i t h d a t a of similar c o m p o u n d s f r o m earlier s t u d i e s g, x~ 13, t h e s t r u c t u r e s of t h e c o m p o u n d s
EXPERIENTIA 33/9
s h o w n i n t h e S c h e m e could b e d e t e r m i n e d . I n all cases f i n a l p r o o f was o b t a i n e d b y s y n t h e s i s . B e c a u s e t h e comp o u n d s 3 b , 3 c a n d 3 d are m a j o r m e t a b o l i t e s , w h i l s t 3 a , 3 e , 3 f a n d 3 g r e p r e s e n t o n l y a few p e r c e n t of t h e t o t a l a m o u n t of m e t a b o l i t e s formed, m o s t likely 3 e, 3 f a n d 3 g were f o r m e d v i a r e d u c t i v e d e c h l o r i n a t i o n of 3 b , 3 c a n d 3 d r e s p e c t i v e l y , since t h e l a t t e r all h a v e a h y d r o x y g r o u p o r t h o to a c h l o r i n e a t o m . T h e p r e s e n t d a t a h o w e v e r , do n o t rule o u t f o r m a t i o n of 3 e , 3 f a n d 3 g v i a t h e 3', 4'e p o x i d e of 3 a , similar t o 3', 4 ' - e p o x i d a t i o n of 3. T h e r e l a t i v e a m o u n t s of t h e u n c h a n g e d c o m p o u n d s , t h e i r h y d r o x y l a t e d a n d d e c h l o r i n a t e d m e t a b o l i t e s isolated f r o m t h e u r i n e are g i v e n in t h e t a b l e .
B i o s y n t h e s i s of i p h o s p h a t i d y l e t h a n o l a m i n e f r o m l C D P - e t h a n o l a m i n e b y t h e
Golgi
complex
! l i v e r in v i t r o 1
of~rat ....
P a t r i c i a L. C h a n g ~, J e n n i f e r M. Sturgess a n d M. A. Moscarello 3
Research Institute, Departments o/Pathology and Biochemistry, The Hospital/or Sick Children, Toronto (Ontario, Canada), 4 February 1977 Summary. A Golgi-rictl f r a c t i o n f r o m r a t liver h a s b e e n s h o w n to s y n t h e s i z e p h o s p h a t i d y l e t h a n o l a m i n e f r o m C D P e t h a n o l a m i n e in vitro. T h e i m p l i c a t i o n s of t h e e x i s t e n c e of s u c h a p a t h w a y for t h e m e m b r a n e flow h y p o t h e s i s are discussed. T h e b i o s y n t h e s i s of p h o s p h o l i p i d s h a s b e e n s h o w n t o o c c u r in e n d o p l a s m i c r e t i c u l u m 4-s a n d in m i t o c h o n d r i a 7, w h e r e a s t h e Golgi c o m p l e x h a s b e e n r e p o r t e d to lack s o m e of t h e e n z y m e s r e q u i r e d in s u c h b i o s y n t h e s i s 5, 0. H o w e v e r , t h i s r e p o r t p r e s e n t s e v i d e n c e t h a t t h e Golgi-rich f r a c t i o n s from rat liver can synthesize phosphatidylethanolamine f r o m C D P - e t h a n o l a m i n e in vitro, d e m o n s t r a t i n g t h a t a t 1000
=o "~ B00 8
R
d
~ ' - -
1,2-diacyl-sn-glycerol + CDP-ethanolamine* ethanolamine phosphotransferase phosphatidylethanolamine* + CMP When CDP-ethanolamine was radioactively labelled w i t h x*C in t h e e t h a n o l a m i n e m o i e t y , t h e r e c o v e r y of radioactive phosphatidylethanolamine measured the a c t i v i t y of e t h a n o l a m i n e p h o s p h o t r a n s f e r a s e , a n e n z y m e r e s p o n s i b l e for t h e f i n a l s t e p of p h o s p h a t i d y l e t h a n o l a m i n e de n o v o s y n t h e s i s a c c o r d i n g t o t h e K e n n e d y p a t h w a y . Materials and methods. All s u b c e l l u l a r f r a c t i o n s were p r e p a r e d f r o m t h e livers of m a l e W i s t a r r a t s w e i g h i n g 200-230 g. T h e r o u g h m i c r o s o m a l f r a c t i o n s were p r e p a r e d b y D a l l n e r ' s m e t h o d s a n d Golgi-rich f r a c t i o n s b y t h e m e t h o d of Sturgess, e t a l ) . T h e p l a s m a m e m b r a n e frac-
600
~-._= 400
l e a s t t h e t e r m i n a l e n z y m e is p r e s e n t . T h e r o u g h e n d o p l a s m i c r e t i c u l u m s h o w e d s i m i l a r s y n t h e t i c a c t i v i t y while t h e p l a s m a m e m b r a n e s h o w e d n o i n c o r p o r a t i o n of C D P ethanolamine. T h e r e a c t i o n u n d e r s t u d y w a s as follows:
ss S s
S
1
200
2 10 20 Time of incubation
30 rain
3 4
Recovery of radioactive phosphatidylethanolamine from CDPethanolamine vs time of incubation with rough microsomal, Golgirich and plasma membrane fractions of the rat liver. Rough microsomal (R, 232 I~g of protein), Golgi-rich (G, 139 v.g of protein) or plasma membrane (P, 117 p.g of protein) fractions were each incubated at 37 ~ in a medium containing CDP-[14C]-ethanolamine and diglyceride (see Materials and methods) in quadruplicate. At each time interval: 0, 10, 20 and 30 min, incubation was terminated, phospholipids were extracted from the total incubation mixture and separated by TLC. The spot corresponding to phosphatidylethanolamine (PE) was scraped and counted in a liquid scintillation system.
5 6 7 8 9
Acknowledgments. This study was supported by the Medical Research COuncil of Canada. Present address: Department of Paediatrics, McMaster Medical Center, Hamilton, Ontario, Canada. Reprint requests to be addressed to Dr M. A. Moscarello, Research Institute, Hospital for Sick Children, Toronto, M5G 1X8, Ontario, Canada. G . F . Wilgram and E. P. Kennedy, J. biol. Chem. 238, 2615 (1963). L . M . G . Van Golde, B. Fleischer and S. Fleischer, Biochim. biophys. Acta 249, 318 (1971). L.M.G. Van Golde, J. Raben, J. J. Batenburg, B. Fleischer, F. Zambrano and S. Fleischer, Biochim. biophys. Acta 360, 179 (1974). W.C. McMurray and R. M. C. Dawson, Biochem. J. 112, 91 (1969). G. Dallner, in: Methods in Enzymology. Ed. S. P. Colowick and N. O. Kaplan. Academic Press, New York 1974. J.M. Sturgess, E. Katona and M. A. Moscarello, J. Memb. Biol. 12, 367 (1973).
