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Bcitrage zur Okologischen Chemie LXXXIV+
Metabolism of Lower Polychlorinated Biphenyls-'X
in the Rhesus Monkey
by W. Gbeb, W. Klein, F. Coulston*,L. Golderc*. and F. Korte
Institul fur iikologische Chemie tier GeseUschaft fiir
Strahlen tmd Umu'cltforschung mill, Miinchen D-520S St. Augustin 1, If. Germany
*Institute of Experimental Pathology and Toxicology Albany Medical College, Albany, N.Y. 12208
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INTRODUCTION
[
Metabolism studies on PCB are important for estimating j the toxicity of. metabolites after knowing their identii 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, I YOSHIMURA and YAMAMOTO 1973) and pigeons (YOSHIMURA ; and YAMAMOTO 1973). The purpose of this study was to i ' identify all major metabolites after PCB-administration | to Rhesus monkeys and to measure the amounts of diffe' rent excreted metabolites. Correlation ofconversions | and elimination pattern of PCBs with different chlorine ! content might lead to conclusionsabout the behavior of | other PCBs.
I The excretion rates of 2,4'-dichlorobiphenyl and 2,5,2'~trichlotobiphenyl 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'-trichlorobiphenyl, both major components.of the lower AROCLOR series. They were labelled with 4C. Fema le Rhesus monkeys were housed in metabolic cages and the PCB was injected once in the left forearm vein. Three animals received 2,41-dichlorobiphenyl in doses of 16,8, 77.6 and 566/ag/kg, while 82.6/H.g/kg of the 2,5,2'-trichlorobiphenyl were administered to one mon key. Urine and feces were collected daily. Conjugates were hydrolyzed by refluxing urine with an equal volume of 8 N HjSO, *or 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, W., W. Klein, F.COULSTON, L.GOLBERG,and F.KORTE: Bull.Environ.Contam. Toxicol., in press.
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5766 (solvent: CH?C1?) . After methylation by diazomethane
(DE BOER and BACKER 1 963) 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) showed that about 17% of metabolites were conjugated with sulfuric or glucuronic acid. Incubation of urine and feces in vi tro with the original PCBs revealed no formation of meta bolites 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 metaboli tes, no parent compound, were detected in the excreta.
Fig. 1 gives a survey of the metabolites formed from both
PCBs.
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Ri before methyiation {CH2C12)
Rf after methyiation 1 (ch2ci2)
TABLE 1
Metabolites In Monkeys
OH-Metabo- OH-Metabo- CH-Metabo- (CH) --Meta (OH)--Me-
lite
lite S2
lite S3
bolite s4 bol. S5
(OH) _-Me:abof. S6
0.66 0.82
0.50 0.70 .
0.25 0.66
0.13 0.59
< 0.1 0.64
< 0.1 0.52
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GLC after inethylation Rfc (min.)
MS-fragment a|ter methyiation 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.
then temp, then temp, then temp, t hen temp. then temp, 4\ raised 5C raised 5C raised 5C/ raised 5Q raised 5C
mfn.
min.
min.
min.
min.
252 s
252 s
252 s
282 s
282 s
282 s
" M+-CH3 n metastable " M+-C1 " M+-HC1 " M+-CH3-C0
217 1 '_ 209 m
237 1
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217 m
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209 s
237 m 223r m
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247 m 246 m 239 m
267 m -
247 m 246 m 239 1
267 m -
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173 1
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173 m
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139 m ,
204 1
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Peak-Intensities: s=strong, m=medium, l=low
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TABLE 2
TLC-, GLC - and MS-Data of 2,5,2'-Trichlorobiphenyl Metabolites in Monkeys
Data
.
OH-Metabo- OH-Metabo- OH-Metabo- (OH)2-Meta . (OH),-Meta (OH)3-Me-
lite G^
lite G2
lite G3
bolite
bolite G5 tabol. Gg
Rf before methylation (CH2C12)
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0.25
0.25
0.11
<0.1
< 0.1
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0.76
0.72
0.7 2
0.64
0.53
0.32
GLC after methylation Rfc (min.)
MS-fragment a^ter methylation M-*"
" -m+-ch3 " M+-C1 " M+-CH3-CO
13.8/180C
286 s 271 1 251 m 243 m
15.4/185C 16.2/185C 17.0/2O0C 13.7/200C 17.8/2O0C
for 8 min., for 8 min. , for 8'min.,
then temp, then temp, then temp,
raised 5C/ raised 5C/ raised 5C/
min.
min.
min.
286 s 271 1
* 243 1
286 s -
251 1 243 1
316 s 301 1
281 1 273 m
316 s 301 1
273 1
346 s -
-
" M+-CH3-C1
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266 1
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" M+-C12
186 1
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246 1
246 s
276 \
" M+-CH3-C0-C12
173 1
173
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Peak-Intensities: s= strong, m = medium, l=low
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After methylation 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 differed in peak-intensities. All isolated compounds showed the typical isotopic distribution pattern corresponding to the number of chlorine atoms in the parent PCB. For 2,4'-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 should be the precursor of the highest concentrated dihydroxy-compound. Only three monohydroxy-derivatives were formed although six (resp. seven for the trichlorobiphenyl) 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 Bhown in Table 1-2.
Comparing the excreted amounts (in % of totally excreted
radioactivity) of metabolites formed, the dichlorobiphenyl was mainly excreted as monohydroxy-derivatives, while the trichlorobiphenyl was mainly eliminated as di hydroxy-compounds (Table 3).
TABLE 3
Comparison of Metabolites formed of PCBs by Rhesus Monkeys
I
H
es
Metabolites
Dichloro
Trichloro
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biphenyl
biphenyl
H
B
Monohydroxy-metabolites
66.6% .
7.8%
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Dihydroxy-metabolites
32.4%
82.2%
C
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Trihydroxy-metabolites
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6%
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Polar metabolites, unidentified
1% . 4%
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The following might explain this different behavior. Due to the additional chlorine atom, the trichlorobiphenyl is more lipophilic than the dichlorobiphenyl. The mono hydroxy-metabolites react in the same way: the monohydroxy-dichloroblphenyl is excreted rapidly, while the water solubility of the monohydroxy-trichlorobiphenyl is still low. After further hydroxylation the trichlorobiphenyl-metabolites reach a polarity to be easier eli minated. A dihydroxy-trichlorobipheriyl 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
t
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 hydroxylated metabolites and the rapid excre
', iI
tion of the PCBs investigated in primates indicate that accumulation of lower chlorinated biphenyls at low doses
will not occur.
REFERENCES
BLOCK, W.D., DE BOER, Th.
and H.H. CORNISH: J. Biol. Chem. 234, 3301 (1959).
r
J., and H.J.BACKER: Org. Syn. Coll. 4, 943 (1963).
GREB, W., W. KLEIN, F. COULSTON, L. GOLBERG, and F. KORTE; Chemosphere 2, 143 (1973).
GREB, W., W. KLEIN, F. COULSTON, L. GOLBERG, and F. KORTE: Bull. Environ. Contam, Toxicol., in press (1974).
HUTZINGER, 0. et al.: Science 178, 312 (1972).
VOIGT, K.D. in H.D. BERGMEIER: Methods of Enzymatic Analysis. Acad, Press 1965, p. 462.
YOSHIMURA, H., and H. YAMAMOTO: Chem. Pharm. Bull.21,
1168 Cl973) .
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