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it rodxhat J col ier.
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1974). 12
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Bcilrnge zur Okologischcn Chcmie LXXXIV+
Metabolism of Lower Polychlorinated Biphenyls-^C
in the Rhesus Monkev .
*
by W. Grcd, \V. Ki.kin. F. Om;i.sio.n*,L. Goi.iikhc;*, and F: Kortf
lilstitiit furvkvlofifrhe C.bemie tier Cetrllteluijt fiir Strahltn uml l /iueltjtntchiing mbit, Mtinclien
D-S20S St. Aiignnin 1, If. Cennany * Iintitule of Experimental i'utlmlopy and Trn icnlogy
Albany bledical College, Albany, A'.V. 12208
f '
EXHIBIT C
INTRODUCTION
Metabolism studies on PCB are important for estimating the toxicity of. metabolites after knowing their identi ty and biological pathway. Some pure PCB-isomers were
shown to be converted to hydroxylated products by rat liver microsomes (GREB et al. 1974), rabbits {BLOCK and CORNISH 1959), rats (HUTZINGER et al. 1972, YOSHIMURA and YAMAMOTO 1973) and pigeons (YOSHIMURA and YAMAMOTO 1973). The purpose of this study was to Identify all major metabolites after PCB-adminlstratlon to Rhesus monkeys and to measure the amounts of diffe rent excreted metabolites. Correlation of conversions and elimination pattern of PCBs with different chlorine content might lead to conclusions about the behavior of other PCBs.
The excretion rates of 2,4*-dichlorobiphenyl and 2,5,2'-trichloroblphenyl have been described elsewhere (GREB et al. 1973); we now wish to report the identifi cation of their metabolites.
EXPERIMENTAL
.
The PCBs investigated were 2,4'-dichlorobiphenyl and 2,5,2*-trichlo*robiphenyl, both major components .of the lower AROCLOR series. They were labelled with A,C. Fema le Rhesus monkeys were housed in metabolic cages and the PCB was Injected once in the left forearm vein. Three animals received 2,4'-dichlorobiphenyl in doses Of 16.8, 77.6 and 566/ag/kg, while 82.6>ttg/kg of the 2,5,2'-trichlorobiphenyl were administered to one mon ' key. Urine and feces were collected dally. Conjugates were hydrolyzed by refluxing urine with an equal volume of 8 N H2SO. for one hour and extracted with ether. Fe ces were^mixed with anhydrous sodium-sulfate and extrac
ted for two days in a Soxhlet apparatus with CH^Cl, and then with methanol. The combined extracts were concen-
+LXXXIII. Communication GREB, K., W. Klein, F.COULSTON,' L.GOLBERG,and F.KORTE: Bull.Environ.Contam.
. Toxicol., in press.
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trated under a nitrogen stream and purified by prepara tive layer chromatography on silica gel Merck Nr.5765, . 5766(solventj CH2CI5) After methylation by diazomethane (DE BOER and BACKER 1963) the metabolites were characte
rized by GLC (Packard 873, EC-detector, 1% OV-1, 2m) and identified by GLC-MS (LKB 9000 A).
RESULTS AND DISCUSSION '
After 14 days about 77% of the administered radioactivity
were recovered,and reaction of urine with Glusulase (Endo `
Laboratories) according to K.D, VOIGT (1965) show'ed that
' about 17% of metabolites were conjugated with Bulfuric
'
or glucuronic *cl&. Incubation of urine and feces in vi
tro with the original PCBs revealed no formation of meta-'
bolltes due to reaction with bacteria from the intestines.
In vivo urinary and fecal metabolites were identical and" the metabolic pattern in excreta was constant between ;
the first and fifth day after application. Only metabolites, no parent compound, were detected in the excreta. .>
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,
Pig. 1 gives a survey of the metabolites formed from both
PCBs.
. ..
Pig.1.Metabolism of Polychlorinated Biphenyls by
Rhesus Monkeys
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. TABLE 1
TLC Data
GLC- and MS-Data of 2,4*-Dichloroblphenyl Metabolites In Monkeys
CH-Metabo- OH-Metabo- CH-Metabo- (CH)--Meta "(OH) ,-Me-
lite Sj
lite S2
lite S3
bolite S4 bol. S5
Rf before methylation 1 'ch2ci2)
0.66
0.50
0.25
0.13
< 0.1
R< after methylation * !CH2C12)
0753
o77o
0.66
0.59
. 0.64
!CH),-Me:aboI.
< 0.1 0.52
GLC after methylation P-t (min.)
MS-fragment a|ter methylation M
10.2/179C 12.1/185C 11.4/185C 14.8/200C 13.1/200C 11.6/200C
for 9 min. for 9 min. for 8 min., for 8 min. for 8 min.r
then temp, then temp, then temp, then.temp, then temp,
raised 5C raised 5C raised 5C/ raised 5Q raised 5C/
min.
min.
min.
min.
! min.
252 s
252 s
252 s
282 s
282 s | 282 s
M+-CH3 ' metastable * M+-C1 * M+-HC1 * m+-ch3-co
217 1 -
209 m
237 1
217 m
-
209 s
237 m 223* m
-
-- 209 m
267 1 -
247 m 246 m 239 m
267 m -
247 m 246 m 239 1
j 267 m "
_
239 1
1
1 w i
o u>
' m+-ch3-ci
- - 202 1 - - -
se
" m+-ch3-co-hci
173 1 - . .173 m
-
-
'.
o (jj
_ ' M+-CH3-C0-C12
-
- . 139 m
204 1
rv
O' Feax-Intensities: sstrong, m*=medium, llow
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TABLE 2
TLC-, GLC - and MS-Data of 2,5,2 *-Trichlorobiphenyl Metabolites in Monkeys
Data
OH-Metabo OH-Ketabo- OH-Metabo- (OH)--Meta . (OH)--Meta '(OH)--Me-
lite
lite G2
lite G3
bolite G^ bolite g5 tabol. Gg
Rf before methylation r (ch2ci2)
0.49
0.25
0.25
0.11
<0.1
< 0.1
after methylation' * (ch2ci2)
0.76
0.72
0.72
0.64
0.53
0.32
GLC after methylation R^. (min.)
MS-fragnent ater methylation M`
" -M+-CH3
" M+-C1 " M+-CH3-CO
13.8/180C
286 s 271 1 251 m 243 m
1S.4/185C 16,2/185C 17,0/20OC 13.7/200C 17.8/200C
for 8 min., for 8 min., for 8 min.
then temp, then temp, then temp,
raised 5C/ raised 5C/ raised 5C
min.
rain.
min.
286 s 271 1
-
243 1
286 s -
251 1 243 1
316 s 301 1
281 1 273 m
316 s301 1
273 1
346 s -
j-
" M+-CH3-Cl
-
- '-
266 1
-.
-
" M+-Cl2
186 1
-
-
246 1
246; s
1 276 1
* M+-CH3-C0-C12
173 1
173
- 203 1
-
-
Peak-Intensities* s strong, m * medium, l*low
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After inethylation all purified metabolites showed in the
mass spectra -OCH,-groups Indicating that the original
metabolites were phenolic derivatives. In both cases
a number of isomers had been formed. Isomers only dif
fered in peak-intensities. All isolated compounds showed
the typical isotopic distribution pattern corresponding
to the number of chlorine atoms in the parent PCB. For
2,41-dichlorobiphenyl there was no conversion exceeding
the Introduction of 2 OH-groups per molecule. For the
2,5,2,-trichlorobiphenyl we observed a metabolite with
three OH-groups. Corresponding to its formation the
monohydroxy-metabolite with the lowest concentration , .
Bhould be the precursor of the highest concentrated di
hydroxy-compound. Only three monohydroxy-derivatives .
were formed although six (resp. seven for the trichloro-
biphenyl) structures are possible. For each PCB a highly
polar zone of 1 and 4% resp. of radioactivity could not
be identified. Data of the metabolites are shown in '
Table 1-2.
.
Comparing the excreted amounts (in % of totally excreted
radioactivity) of metabolites formed, the dichlorobl-
phenyl was mainly excreted as monohydroxy-derivatives,
while the trlchlorobiphenyl was mainly eliminated as di-
hydroxy-compounds (Table 3).
; v.
TABLE 3 '
.. .
`
Comparison of Metabolites formed of PCBs by Rhesus
Monkeys
. r;
.
Metabolites
* Dichloro- . Trichloro-
biphenyl
biphenyl
Monohydroxy-metabolites
Dihydroxy-metabolites
Trihydroxy-metabolites
Polar metabolites, unidentified
.
66.6% 32.41
- ' 1%
7.6% . 82.21
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The following might explain this different behavior. Due
to the additional chlorine atom, the trichlorobiphenyl
ie more lipophilic than the dichloroblphenyl. The mono
hydroxy-metabolites react in the same way: the mono-
hydroxy-dichlorobiphenyl is excreted rapidly, while the
water solubility of the monohydroxy-trichlorobiphenyl
is still low. After further hydroxylation the trichloro-
biphenyl-metabolites reach a polarity to be easier eli
minated. A dihydroxy-trichlorobiphenyl thus should
correspond to a monohydroxy-dichlorobiphenyl as regards
speed of elimination. For higher chlorinated BPs, a
higher degree of hydroxylation is necessary and will
cause a longer retention time of the compound in the bo
dy, that is, a slow excretion rate. This might explain
their accumulation.
..
CONCLUSION
Metabolism of PCB with high chlorine content will be dif
ficult and proceed slowly. Concerning lower chlorinated
biphenyls, however, this study reveals that total degra
dation to hydroxyloted metabolites and the rapid excre
tion of the PCBs investigated in primates Indicate that
accumulation of lower chlorinated biphenyls at low doses
will not occur.
,
REF-E'*>RE...N..CES
BLOCK, W.D., and H.H.^OCNISH? J Biol. Chem. 234, 3301 (1959Jj
DE BOER, Th. J., and tf^BACKER: Org Syn. Coll. 4 , 943 U&3) .
GREB, W., W. KLEIN, F. COULSTON, L. GOLBERG, and F. KORT^Chemosphere 2, 143 (1973).
c.m:B, W., W. KLEIN, i-***3puI.f;WN, L. GOLRERG, and F. KORTE^I'ull. Environ. Contarn. Toxicolpress (1974).
HUTZINGER, 0. et al.y^ience 178, 312 (1972).
VOIGT, K.D. in H.D. RE&gMEIER: Methods of Enzymatic AnalyslffvoAcad. Press 1965, p. 462.
YOSHIMURA, H., and H^AMAMOTO: Chem. Pharm. Bull'. 21.,
1168 (197
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