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Biotransformation of pentachloronitrobenzene-14C in rhesus monkeys after single and chronic oral administration
Chemosphere No. 2, pp 97 - 105. ©Pergamon Press Ltd. 1979. Printed in Gre~t Britain.
BIOTRANSFORMATION
O045-6535/7q/OPOl-O097~O?.OO/O
OF PENTACHLORONITROBENZENE-14C
IN RHESUS MONKEYS
AFTER SINGLE AND CHRONIC ORAL ADMINISTRATION W. K~gel, W.F. MUller,
F. Coulston +, F. Korte
Institut f~r Chemie der Technischen
Universit~t
D-8050 Freising-Weihenstephan Gesellschaft
fur StrahlenInstitut
und Umweltforschung f~r ~kologische
P.O. Box 1027, Holloman AFB, N.M.
MUnchen
Germany and mbH M~nchen
Chemie 88330 USA
+Albany Medical College International
Center of Environmental
Introduction Residues of Pentachloronitrobenzene dairy products,
(PCNB) have been found in vegetables I,
oils and fats 2. PCNB is the active ingredient
cides used in agriculture order to evaluate possible fate in rhesus monkeys. PCNB in rhesus monkeys findings
Safety, Holloman AFB, N.M. USA
concerning
consequences
Uptake,
for seed - and soil treatment.
is described
storage,
in dogs. The same metabolites and pentachlorobenzene
of PCNB in the non-human primate.
as PCNB metabolites
residues
Kuchar 5 and
(PCTA) as PCNB metabolites group in rats and
of hexachlorobenzene
(QCB) were found, which probably
to the HCB and QCB content of the technical PCNB
(PCA) and N-acetyl-
in rabbits.
were also found by Borzelleca's
studied by that group,
these studies. Using pure radiolabeled
sus monkeys a) after a single oral dose of 2 mg/kg bodyweight
(HCB)
can be attributed mainly
(Terrachlor,
Olin) used for
PCNB, we studied the pattern of metabolites
97
of
elsewhere 3. In this paper we present our
conversion
Borzelleca 6 described PCA and pentachlorothioanisole cows 6. In all animals
and excretion
Betts et al 4 found pentachloroaniline
-L-cystein
In
of PCNB uptake by humans, we studied its
body distribution,
the metabolic
In earlier studies, S-pentachlorophenol
and horticulture
of several fungi-
in rhe-
in six males and two females
9S
No. 2
b) after a single oral dose of 91 mg/kg bodyweight in one female c) during chronic feeding of 2 ppm in the daily diet for 71 days in two males and two females. Materials and Methods Pentachloronitrobenzene, by column chromatography
obtained from ICN Pharmaceuticals Co, was purified
(silica gel/n-hexane).
Electron-capture gas chromato-
graphy was used to establish the absence of hexachlorobenzene or any other chlorinated impurities.
Radiochemically pure 14C-labeled PCNB was obtained from the
Institut f~r 5kologische Chemie der Gesellschaft f~r Strahlen- und Umweltforschung mbH Munich, Germany. Rhesus monkeys
(macaca mulatta) were obtained from
the stock colony of the International Center of Environmental Safety. They were kept in stainless steel metabolism cages (Harford Metal);
feces and urine were
collected daily. Radioactivity in urine was determined by direct liquid scintillation counting (Packard Tri-Carb 2424) using a cocktail of I00 ml methanol and 8 g Permablend III (Packard) in 900 ml dioxane. To determine radioactivity in feces, blood, and organ tissues, samples of 50 - 200 mg were combusted in a Packard Tri-Carb 306 Sample Oxidizer for subsequent liquid scintillation counting For extraction of radioactivity,
the urine was brought to pH 2 with sulfuric
acid and extracted for 24 - 48 hours with ethyl ether in a liquid extractor. Feces were ground with anhydrous sodium sulfate and extracted for 24 h in a soxhlet extractor with methanol. For quantitation of PCNB and metabolites,
aliquots
of the crude extracts were analyzed by radio-thin layer chromatography, using Berthold thin-layer scanners. The radioactive components of the extracts were isolated and purified by combined thin-layer - and column chromatography. A Finnigan-3000 D gaschromatograph-mass metabolites
spectrometer was used for identification of
(column: 5' x 2 m m i.d. 3 % OV i on Supelcoport).
Results and Discussion The urinary and fecal extracts from the three experiments: a) single oral dose, 2mg/kg b) single oral dose, 91 mg/kg c) chronic feeding, 2 ppm of daily diet were prepurified by column chromatography,
using silica gel and 400 ml each of
benzene/chloroform i.: i (v/v) chloroform acetonitrile methanol The radioactive fractions were further purified by thin-layer chromatography on preparative TLC plates. In experiment a), a mixture of i0 % acetone in n-hexane was used to develop the chromatograms,
in the experiments b) and c) pure hexane
was used as a solvent. The R -values and the percentage distribution of radioactive zones found in the three experiments are listed in table I.
No. P_
99
TABLE I Rf-Values and Percentage Distribution of Radioactive Zones Separated by TLC of Urine - and Feces Extracts Experiment a
b
c
Solvent hexane/acetone 9:1
hexane
hexane
Urine Rf 0.00-0.12 0.64-0.83 0.83-1.00
Feces 21.3 55.2 23.5
0?.E0-U. I z 0.12-0.26 0.26-0.39 0.39-0.53 0.53-0.75 0.00-0.26 0.26-0.52 0.52-0.62 0.62-0.77
19.3 23.2 22.4 10.6 24.5 49.0 29.3 11.9 9.8
%
Rf 00-0.16 35-0.55 55-0.63 69-0.87 UU-U.U~ 08-0.32 32-0.45 45-0.60
% 33.6 42.0 5.2 19.2 l.u 51.3 25.9 15.8
0.00-0.20 0.20-0.47 0.47-0.58 0.58-0.76
15.9 38.2 32.4 13.5
0 0 0 0 O 0 0 0
The radioactive zones were removed from the plates, and washed with methanol; the concentrated eluates were analyzed by GLC-MS. The metabolites were characterized by their respective retention times and mass spectra and compared to authentic synthesized compounds of proven structure, as far as these were available. Table II lists PCNB and 7 metabolites indentified in this way. TABLE II Chemical Structures, Retention Times, and Mass Metabolite Structure 293 ParentPentachlorocompound nitrobenzene 247 CI~CI
CI/'~L_--"~CI Cl
I
Pentachloroaniline
NH 2 CI~CI CI~,~CI Cl
Spectra of PCNB Metabolites I-VII Mass Spectrum Rt(min) ~ (M+), 263 (M+-NO) 4.2
235 212 200 177 142 107
(M+-N02) (M+-CN02) (M+-N02-CI) (M+-CN02-CI) (M+-N02-2CI) (M+-N02-3CI) (M+-N02-4CI)
263 227 201 193 192 165 130 95
(M+), 236 (M+-HCN) (M+-HCI) (M+-HCN-CI) (M+-2CI) (M+-CI-HCI) (M+-HCN-HCI-CI) (M+-2CI-HCN-HCI) (M+-4CI-HCN)
4.5
I00
I~o. 2
Table II - cont. Metabolite
II
Pentachlorobenzene
H CI-~Cl CI/~/~'CI Cl
III
Pentachlorophenol
OH CI~CI
CI/"~Cl C] IV
Pentachlorothioanisole
SCH3 CI~Cl C l / ~ - Cl Cl
V
Pentachlorothiophenol
SH CI~Cl Cl" " , , . ~ CI Cl
Vl
2,3,4,5Tetrachloroaniline
NH2 H,]~CI
Cl VII
2,3,5,6Tetrachlorophenol, methylated
OH CI~Cl H
* 160°C
Mass Spectrum
Structure
Rt(min) 1.3
248 178 143 108
(M+), 213 (M+-CI) (M+-2CI) (M+-3CI) (M+-4CI)
264 200 165 130 95
(M+), 235 (M+-COH) (M+-COH-CI) (M+-COH-2CI) (M+-COH-3CI) (M+-COH-4CI)
2.9
294 261 259 244 209 174 139
(M+), 279 (M+-CH3) (M+-SH) (M+-CI) (M+-CH3-CI) (M+-CH3-2CI) (M+-CH3-3CI) (M+-CH3-4CI)
6.2
280 (M+) 245 (M+-CI) 210 (M+-2CI)
2.5
299 202 166 157 131
(M+) (M+-HCN) (M+-CNH2-CI) (M+-2HCI) (M+-CNH2-2CI)
3.0
244 213 209 201 166 131 96
(i'4+),229 (M+-CH3) (M+-OCH3) (M+-CI) (M+-CH3-CO) (M+-CH3-CO-CI) (M+-CH3-CO-2CI) (M+-CH3-CO-3CI)
1.4
No.
2
I01
Another 6 metabolites were characterized by retention times and mass spectra Table III). So far, the proposed structures could not be verified by comparison with authentic synthesized compound~,since these were not available in our laboratory. TABLE III Proposed Structures, Retention Times and Mass Spectra of PCNB Metabolites VlII-XIII Metabolite Structure Mass Spectrum Rt(min)* VIII
Tetrachlorothioanisole
SCH3
CI4 IX
aminothio-Tetrachl°r°anisole
NH2
H3CS X
Tetrachloroaminophenylmethylsulfoxide
ClL
NH
260 227 225 210
(M+), 245 (M+-CH 3) (M+-SH) (M+-CI) (M+-CI-CH 3)
3.2
275 (M+) , 260 (M+-CH3) 242 (M+-SH) 225 (M+-CH3-CI)
8.3
291 (M+) 276 (M+-CH3)
7.3
276 (M+) 261 (M+-CH 3) 241 (M+-CI)
13.4
287 (M+) 272 (M+-CH3)
12.5
H3COS~_/IJ-CI4 XI
XII
Tetrachlorophenylmethylsulfoxide
Bis-methylmercaptoaminotrichlorobenzene
c16H3
NH2 H3CS-~ CI3 H3CS~..I
102
No. 2
Table III - cont. Metabolite XIII
Mass Spectrum
Struc tur e Bis-methylmercaptotetrachlorobenzene
SCH3 H3CS-~CIA
Rt(min)* 12.4
306 (M+), 291 (M+-CH3) 276 (M+-2CH3) 271 (M+-CI) 256 (M+-CH3-CI)
* 160Oc **180Oc Tables IV, V, and VI show the percentages of the identified metabolites in urinary and fecal extracts from experiments a, b, and c. The major metabolites are pentachloroaniline, pentachlorobenzene, pentachlorophenol, pentachlorothioanisole, and bis-methylmercapto-tetrachlorobenzene. It is remarkable that the pattern of metabolites was very similar after the low single oral dose and the low-dose chronic exposure. After the high single oral dose, the relative amount of pentachlorophenol was increased at the expense of pentachloroaniline. Also, metabolites VII-XII and 5 more unidentified metabolites were found in this experiment, which were not detected after low-dose exposure. Failure to find these metabolites in experiments a and c may, however, be due to the low concentrations which would be expected in urine and feces after the uptake of small amounts of PCNB. TABLE IV Percentages of PCNB and Metabolites in Extracts of Urine and Feces After a Single Oral Dose of 2 m~/k5 Metabolite
Structure
% of urinary extract
I
Pentachloroaniline
55.2
II
Pentachlorobenzene
11.7
Pentachlorophenol
12.2
III IV
Pentachlorothioanisole
V Vl Xlll IX
66.0 1.0
9.7
6.2
Pentachlorothiophenol
1.4
0.2
2,3,4,5-Tetrachloroaniline
0.I
0.i
9.7
7.1
Bis-methylmercaptotetrachlorobenzene Tetrachloroaminothioanisole
Parentcompound
% of fecal extract
Pentachloronitrobenzene
3.1
16.3
No. 2
103
TABLE V Percentages of PCNB and Metabolites in Extracts of Urine and Feces After a Single 0ral Dose of 91 mg/kg Metabolite
Structure
I II III IV V Vl Vll Vlll IX X
Pentachloroaniline Pentachlorobenzene Pentachlorophenol Pentachlorthioanisole Pentachlorothiophenol 2,3,4,5-Tetrachloroaniline 2,3,5,6-Tetrachlorophenol Tetrachlorothioanisole Tetrachloroaminothioanisole Tetrachloroaminophenylmethylsulfoxide Tetrachlorophenylmethylsulfoxide Bis-methylmercapto-aminotrichlorobenzene Bis-methylmercapto-tetrachlorobenzene
XI XII Xlll XV XVl XVll XVIII XlX XX Parentcompound
% of urinary extract
Pentachlorophenylmethylsulfoxide
% of fecal extract
36.0 11.9 17.5 10.3 1.4 1.0 1.8 3.4 1.6
66.2 i.i
0.3
2.0
6.2 0.2 0.I 3.1
3.7 0.I 9.2 0.01 0.i 0.67 0.01
7 .i i.i
0.01 1.0
Pentachloronitrobenzene
12.9
TABLE VI Percentages of PCNB and Metabolites in Extracts of Urine and Feces During Metabolite I II III IV V Vl XIII IX Parentcompound
Chronic Feedin~ of 2 ppm in the Daily Diet Structure % of urinary extract Pentachloroaniline Pentachlorobenzene Pentachlorophenol Pentachlorothioanisole Pentachlorothiophenol 2,3,4,5-Tetrachloroaniline Bis-methylmercapto-tetrachlorobenzene Tetrachloroaminothioanisole Pentachloronitrobenzene
55.4 11.8 12.2 9.8 1.0 0.i 9.7
% of fecal extract 70.6 0.5 6.0 0.i 0.01 9.3 0.09 13.4
The bile obtained from sacrificed animals in experiments a and c contained highly polar radioactive material which was analyzed by thin layer chromatography on silica gel, using a solvent system of 65 parts chloroform, 35 parts methanol and i0 parts water. The Rf-values and percentages are given in Table VII
104
No. 2
TABLE Vll Rf-Values and Percentages of Biliary Metabolites Zone
Rf-value
B B B B B
0.00-0.12 0.12-0.28 0.28-0.48 0.52-0.58 0.91-1.00
i 2 3 4 5
% 27.5 18.8 32.7 15.6 5.4
Zone B 5 contained mainly pentachloroaniline; small amounts of metabolites II, IV, and VI, were present. The thin-layer chromatographic behavior of the polar metabolites suggests that they are conjugates. Treatment with 9N ~CI at 60°C for 24 hours or incubation with glucuronidase/sulfatase did not produce less polar derivatives, so it is unlikely that these fractions contain glucuronide or sulfate conjugates. The assumption that the polar biliary metabolites are mercapture acids is supported by the presence of sulfur-containing metabolites in the feces which are probably breakdown products of thioaminoacid conjugates. Conclusion The two major pathways of biotransformation of pentachloronitrobenzene in the rhesus monkey are a) the reduction of the nitro-moiety to the corresponding aniline, and b) the cleavage of the C-N bond, presumably via conjugation with sulfur-containing amino acids, e.g. glutathione. Subsequent metabolic breakdown of these conjugates can produce pentachlorothioanisole, pentachlorothiophenol, pentachlorophenol or pentachlorobenzene. The conjugation can occur twice with one molecule, resulting in bis-methylmercapto-tetrachlorobenzene and other tetrachlorometabolites without nitrogen, or in combination with nitro-reduction, leading to tetrachloro-amino-thioanisole, tetrachloroaniline, tetrachloro-aminophenol, or -aminothiophenol. Some of the numerous possible metabolites were found only in small quantities after a relatively large dose of PCNB, but pentachloroaniline, pentachlorophenol, pentachlorobenzene, pentachlorothioanisole, and bis-methylmercapto-tetrachlorobenzene were always present as major metabolites A similar pattern of metabolites in rats 7 shows that the pathways of biotransformation found in this study are not unique in the rhesus monkey. The earlier results of Borzelleca's group who found only pentachloroaniline,
-thioanisole,
and -benzene do not contradict our findings; since they used unlabeled PCNB and extracted the organs and excreta of their test animals only with hexane, the more polar metabolites probably escaped their analysis. The multitude of available mechanisms to convert PCNB to more polar metabolites explains the rapid excretion and extremely low storage tendency of this chemical in mammals.
No. 2
105
Acknowledgement This work was carried out within the frame of a cooperative research project between the Albany Medical College with International Center of Environmental Safety, Alamogordo, N.M. and the Society for Radiation and Environmental Research, Munich, on "Ecologic-Toxicologic Effects of Foreign Compounds in NonHuman Primates and Other Laboratory Animals", which is sponsored by the Department of Health, Education and Welfare as well as the Environmental Protection Agency of the United States of America and by the Federal Ministry of Research and Technology of the Federal Republic of Germany. References i
W. Dejonckheere, W. Steurbaut, R.H. Kips - Pestic. Monit. J. I0, 68 (1976)
2
P.E. Corneliussen,
3
W. K8gel, W.F. MUller, F. Coulston, F. Korte - Chemosphere in press
- Pestic. Monit. J. 2, 140 (1969)
4
J.J. Betts, S.P. James, W.V. Thorpe - Biochem. J. 61, 611 (1955)
5
E.J. Kuchar, F.O. Geenty, W.P. Griffith, R.J. Thomas - J. Agr. Food Chem.
6
17, 1237 (1969) J.F. Borzelleca, P.S. Larson, E.M. Crawford, G.R. Hennigar Jr., E.J. Kuchar, H.H. Klein - Toxicol. Appl. Pharmacol.
7
M. Volz - priv. communication (Received in Germany 28 December 1978)
18, 522 (1971)
BIOTRANSFORMATION
O045-6535/7q/OPOl-O097~O?.OO/O
OF PENTACHLORONITROBENZENE-14C
IN RHESUS MONKEYS
AFTER SINGLE AND CHRONIC ORAL ADMINISTRATION W. K~gel, W.F. MUller,
F. Coulston +, F. Korte
Institut f~r Chemie der Technischen
Universit~t
D-8050 Freising-Weihenstephan Gesellschaft
fur StrahlenInstitut
und Umweltforschung f~r ~kologische
P.O. Box 1027, Holloman AFB, N.M.
MUnchen
Germany and mbH M~nchen
Chemie 88330 USA
+Albany Medical College International
Center of Environmental
Introduction Residues of Pentachloronitrobenzene dairy products,
(PCNB) have been found in vegetables I,
oils and fats 2. PCNB is the active ingredient
cides used in agriculture order to evaluate possible fate in rhesus monkeys. PCNB in rhesus monkeys findings
Safety, Holloman AFB, N.M. USA
concerning
consequences
Uptake,
for seed - and soil treatment.
is described
storage,
in dogs. The same metabolites and pentachlorobenzene
of PCNB in the non-human primate.
as PCNB metabolites
residues
Kuchar 5 and
(PCTA) as PCNB metabolites group in rats and
of hexachlorobenzene
(QCB) were found, which probably
to the HCB and QCB content of the technical PCNB
(PCA) and N-acetyl-
in rabbits.
were also found by Borzelleca's
studied by that group,
these studies. Using pure radiolabeled
sus monkeys a) after a single oral dose of 2 mg/kg bodyweight
(HCB)
can be attributed mainly
(Terrachlor,
Olin) used for
PCNB, we studied the pattern of metabolites
97
of
elsewhere 3. In this paper we present our
conversion
Borzelleca 6 described PCA and pentachlorothioanisole cows 6. In all animals
and excretion
Betts et al 4 found pentachloroaniline
-L-cystein
In
of PCNB uptake by humans, we studied its
body distribution,
the metabolic
In earlier studies, S-pentachlorophenol
and horticulture
of several fungi-
in rhe-
in six males and two females
9S
No. 2
b) after a single oral dose of 91 mg/kg bodyweight in one female c) during chronic feeding of 2 ppm in the daily diet for 71 days in two males and two females. Materials and Methods Pentachloronitrobenzene, by column chromatography
obtained from ICN Pharmaceuticals Co, was purified
(silica gel/n-hexane).
Electron-capture gas chromato-
graphy was used to establish the absence of hexachlorobenzene or any other chlorinated impurities.
Radiochemically pure 14C-labeled PCNB was obtained from the
Institut f~r 5kologische Chemie der Gesellschaft f~r Strahlen- und Umweltforschung mbH Munich, Germany. Rhesus monkeys
(macaca mulatta) were obtained from
the stock colony of the International Center of Environmental Safety. They were kept in stainless steel metabolism cages (Harford Metal);
feces and urine were
collected daily. Radioactivity in urine was determined by direct liquid scintillation counting (Packard Tri-Carb 2424) using a cocktail of I00 ml methanol and 8 g Permablend III (Packard) in 900 ml dioxane. To determine radioactivity in feces, blood, and organ tissues, samples of 50 - 200 mg were combusted in a Packard Tri-Carb 306 Sample Oxidizer for subsequent liquid scintillation counting For extraction of radioactivity,
the urine was brought to pH 2 with sulfuric
acid and extracted for 24 - 48 hours with ethyl ether in a liquid extractor. Feces were ground with anhydrous sodium sulfate and extracted for 24 h in a soxhlet extractor with methanol. For quantitation of PCNB and metabolites,
aliquots
of the crude extracts were analyzed by radio-thin layer chromatography, using Berthold thin-layer scanners. The radioactive components of the extracts were isolated and purified by combined thin-layer - and column chromatography. A Finnigan-3000 D gaschromatograph-mass metabolites
spectrometer was used for identification of
(column: 5' x 2 m m i.d. 3 % OV i on Supelcoport).
Results and Discussion The urinary and fecal extracts from the three experiments: a) single oral dose, 2mg/kg b) single oral dose, 91 mg/kg c) chronic feeding, 2 ppm of daily diet were prepurified by column chromatography,
using silica gel and 400 ml each of
benzene/chloroform i.: i (v/v) chloroform acetonitrile methanol The radioactive fractions were further purified by thin-layer chromatography on preparative TLC plates. In experiment a), a mixture of i0 % acetone in n-hexane was used to develop the chromatograms,
in the experiments b) and c) pure hexane
was used as a solvent. The R -values and the percentage distribution of radioactive zones found in the three experiments are listed in table I.
No. P_
99
TABLE I Rf-Values and Percentage Distribution of Radioactive Zones Separated by TLC of Urine - and Feces Extracts Experiment a
b
c
Solvent hexane/acetone 9:1
hexane
hexane
Urine Rf 0.00-0.12 0.64-0.83 0.83-1.00
Feces 21.3 55.2 23.5
0?.E0-U. I z 0.12-0.26 0.26-0.39 0.39-0.53 0.53-0.75 0.00-0.26 0.26-0.52 0.52-0.62 0.62-0.77
19.3 23.2 22.4 10.6 24.5 49.0 29.3 11.9 9.8
%
Rf 00-0.16 35-0.55 55-0.63 69-0.87 UU-U.U~ 08-0.32 32-0.45 45-0.60
% 33.6 42.0 5.2 19.2 l.u 51.3 25.9 15.8
0.00-0.20 0.20-0.47 0.47-0.58 0.58-0.76
15.9 38.2 32.4 13.5
0 0 0 0 O 0 0 0
The radioactive zones were removed from the plates, and washed with methanol; the concentrated eluates were analyzed by GLC-MS. The metabolites were characterized by their respective retention times and mass spectra and compared to authentic synthesized compounds of proven structure, as far as these were available. Table II lists PCNB and 7 metabolites indentified in this way. TABLE II Chemical Structures, Retention Times, and Mass Metabolite Structure 293 ParentPentachlorocompound nitrobenzene 247 CI~CI
CI/'~L_--"~CI Cl
I
Pentachloroaniline
NH 2 CI~CI CI~,~CI Cl
Spectra of PCNB Metabolites I-VII Mass Spectrum Rt(min) ~ (M+), 263 (M+-NO) 4.2
235 212 200 177 142 107
(M+-N02) (M+-CN02) (M+-N02-CI) (M+-CN02-CI) (M+-N02-2CI) (M+-N02-3CI) (M+-N02-4CI)
263 227 201 193 192 165 130 95
(M+), 236 (M+-HCN) (M+-HCI) (M+-HCN-CI) (M+-2CI) (M+-CI-HCI) (M+-HCN-HCI-CI) (M+-2CI-HCN-HCI) (M+-4CI-HCN)
4.5
I00
I~o. 2
Table II - cont. Metabolite
II
Pentachlorobenzene
H CI-~Cl CI/~/~'CI Cl
III
Pentachlorophenol
OH CI~CI
CI/"~Cl C] IV
Pentachlorothioanisole
SCH3 CI~Cl C l / ~ - Cl Cl
V
Pentachlorothiophenol
SH CI~Cl Cl" " , , . ~ CI Cl
Vl
2,3,4,5Tetrachloroaniline
NH2 H,]~CI
Cl VII
2,3,5,6Tetrachlorophenol, methylated
OH CI~Cl H
* 160°C
Mass Spectrum
Structure
Rt(min) 1.3
248 178 143 108
(M+), 213 (M+-CI) (M+-2CI) (M+-3CI) (M+-4CI)
264 200 165 130 95
(M+), 235 (M+-COH) (M+-COH-CI) (M+-COH-2CI) (M+-COH-3CI) (M+-COH-4CI)
2.9
294 261 259 244 209 174 139
(M+), 279 (M+-CH3) (M+-SH) (M+-CI) (M+-CH3-CI) (M+-CH3-2CI) (M+-CH3-3CI) (M+-CH3-4CI)
6.2
280 (M+) 245 (M+-CI) 210 (M+-2CI)
2.5
299 202 166 157 131
(M+) (M+-HCN) (M+-CNH2-CI) (M+-2HCI) (M+-CNH2-2CI)
3.0
244 213 209 201 166 131 96
(i'4+),229 (M+-CH3) (M+-OCH3) (M+-CI) (M+-CH3-CO) (M+-CH3-CO-CI) (M+-CH3-CO-2CI) (M+-CH3-CO-3CI)
1.4
No.
2
I01
Another 6 metabolites were characterized by retention times and mass spectra Table III). So far, the proposed structures could not be verified by comparison with authentic synthesized compound~,since these were not available in our laboratory. TABLE III Proposed Structures, Retention Times and Mass Spectra of PCNB Metabolites VlII-XIII Metabolite Structure Mass Spectrum Rt(min)* VIII
Tetrachlorothioanisole
SCH3
CI4 IX
aminothio-Tetrachl°r°anisole
NH2
H3CS X
Tetrachloroaminophenylmethylsulfoxide
ClL
NH
260 227 225 210
(M+), 245 (M+-CH 3) (M+-SH) (M+-CI) (M+-CI-CH 3)
3.2
275 (M+) , 260 (M+-CH3) 242 (M+-SH) 225 (M+-CH3-CI)
8.3
291 (M+) 276 (M+-CH3)
7.3
276 (M+) 261 (M+-CH 3) 241 (M+-CI)
13.4
287 (M+) 272 (M+-CH3)
12.5
H3COS~_/IJ-CI4 XI
XII
Tetrachlorophenylmethylsulfoxide
Bis-methylmercaptoaminotrichlorobenzene
c16H3
NH2 H3CS-~ CI3 H3CS~..I
102
No. 2
Table III - cont. Metabolite XIII
Mass Spectrum
Struc tur e Bis-methylmercaptotetrachlorobenzene
SCH3 H3CS-~CIA
Rt(min)* 12.4
306 (M+), 291 (M+-CH3) 276 (M+-2CH3) 271 (M+-CI) 256 (M+-CH3-CI)
* 160Oc **180Oc Tables IV, V, and VI show the percentages of the identified metabolites in urinary and fecal extracts from experiments a, b, and c. The major metabolites are pentachloroaniline, pentachlorobenzene, pentachlorophenol, pentachlorothioanisole, and bis-methylmercapto-tetrachlorobenzene. It is remarkable that the pattern of metabolites was very similar after the low single oral dose and the low-dose chronic exposure. After the high single oral dose, the relative amount of pentachlorophenol was increased at the expense of pentachloroaniline. Also, metabolites VII-XII and 5 more unidentified metabolites were found in this experiment, which were not detected after low-dose exposure. Failure to find these metabolites in experiments a and c may, however, be due to the low concentrations which would be expected in urine and feces after the uptake of small amounts of PCNB. TABLE IV Percentages of PCNB and Metabolites in Extracts of Urine and Feces After a Single Oral Dose of 2 m~/k5 Metabolite
Structure
% of urinary extract
I
Pentachloroaniline
55.2
II
Pentachlorobenzene
11.7
Pentachlorophenol
12.2
III IV
Pentachlorothioanisole
V Vl Xlll IX
66.0 1.0
9.7
6.2
Pentachlorothiophenol
1.4
0.2
2,3,4,5-Tetrachloroaniline
0.I
0.i
9.7
7.1
Bis-methylmercaptotetrachlorobenzene Tetrachloroaminothioanisole
Parentcompound
% of fecal extract
Pentachloronitrobenzene
3.1
16.3
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103
TABLE V Percentages of PCNB and Metabolites in Extracts of Urine and Feces After a Single 0ral Dose of 91 mg/kg Metabolite
Structure
I II III IV V Vl Vll Vlll IX X
Pentachloroaniline Pentachlorobenzene Pentachlorophenol Pentachlorthioanisole Pentachlorothiophenol 2,3,4,5-Tetrachloroaniline 2,3,5,6-Tetrachlorophenol Tetrachlorothioanisole Tetrachloroaminothioanisole Tetrachloroaminophenylmethylsulfoxide Tetrachlorophenylmethylsulfoxide Bis-methylmercapto-aminotrichlorobenzene Bis-methylmercapto-tetrachlorobenzene
XI XII Xlll XV XVl XVll XVIII XlX XX Parentcompound
% of urinary extract
Pentachlorophenylmethylsulfoxide
% of fecal extract
36.0 11.9 17.5 10.3 1.4 1.0 1.8 3.4 1.6
66.2 i.i
0.3
2.0
6.2 0.2 0.I 3.1
3.7 0.I 9.2 0.01 0.i 0.67 0.01
7 .i i.i
0.01 1.0
Pentachloronitrobenzene
12.9
TABLE VI Percentages of PCNB and Metabolites in Extracts of Urine and Feces During Metabolite I II III IV V Vl XIII IX Parentcompound
Chronic Feedin~ of 2 ppm in the Daily Diet Structure % of urinary extract Pentachloroaniline Pentachlorobenzene Pentachlorophenol Pentachlorothioanisole Pentachlorothiophenol 2,3,4,5-Tetrachloroaniline Bis-methylmercapto-tetrachlorobenzene Tetrachloroaminothioanisole Pentachloronitrobenzene
55.4 11.8 12.2 9.8 1.0 0.i 9.7
% of fecal extract 70.6 0.5 6.0 0.i 0.01 9.3 0.09 13.4
The bile obtained from sacrificed animals in experiments a and c contained highly polar radioactive material which was analyzed by thin layer chromatography on silica gel, using a solvent system of 65 parts chloroform, 35 parts methanol and i0 parts water. The Rf-values and percentages are given in Table VII
104
No. 2
TABLE Vll Rf-Values and Percentages of Biliary Metabolites Zone
Rf-value
B B B B B
0.00-0.12 0.12-0.28 0.28-0.48 0.52-0.58 0.91-1.00
i 2 3 4 5
% 27.5 18.8 32.7 15.6 5.4
Zone B 5 contained mainly pentachloroaniline; small amounts of metabolites II, IV, and VI, were present. The thin-layer chromatographic behavior of the polar metabolites suggests that they are conjugates. Treatment with 9N ~CI at 60°C for 24 hours or incubation with glucuronidase/sulfatase did not produce less polar derivatives, so it is unlikely that these fractions contain glucuronide or sulfate conjugates. The assumption that the polar biliary metabolites are mercapture acids is supported by the presence of sulfur-containing metabolites in the feces which are probably breakdown products of thioaminoacid conjugates. Conclusion The two major pathways of biotransformation of pentachloronitrobenzene in the rhesus monkey are a) the reduction of the nitro-moiety to the corresponding aniline, and b) the cleavage of the C-N bond, presumably via conjugation with sulfur-containing amino acids, e.g. glutathione. Subsequent metabolic breakdown of these conjugates can produce pentachlorothioanisole, pentachlorothiophenol, pentachlorophenol or pentachlorobenzene. The conjugation can occur twice with one molecule, resulting in bis-methylmercapto-tetrachlorobenzene and other tetrachlorometabolites without nitrogen, or in combination with nitro-reduction, leading to tetrachloro-amino-thioanisole, tetrachloroaniline, tetrachloro-aminophenol, or -aminothiophenol. Some of the numerous possible metabolites were found only in small quantities after a relatively large dose of PCNB, but pentachloroaniline, pentachlorophenol, pentachlorobenzene, pentachlorothioanisole, and bis-methylmercapto-tetrachlorobenzene were always present as major metabolites A similar pattern of metabolites in rats 7 shows that the pathways of biotransformation found in this study are not unique in the rhesus monkey. The earlier results of Borzelleca's group who found only pentachloroaniline,
-thioanisole,
and -benzene do not contradict our findings; since they used unlabeled PCNB and extracted the organs and excreta of their test animals only with hexane, the more polar metabolites probably escaped their analysis. The multitude of available mechanisms to convert PCNB to more polar metabolites explains the rapid excretion and extremely low storage tendency of this chemical in mammals.
No. 2
105
Acknowledgement This work was carried out within the frame of a cooperative research project between the Albany Medical College with International Center of Environmental Safety, Alamogordo, N.M. and the Society for Radiation and Environmental Research, Munich, on "Ecologic-Toxicologic Effects of Foreign Compounds in NonHuman Primates and Other Laboratory Animals", which is sponsored by the Department of Health, Education and Welfare as well as the Environmental Protection Agency of the United States of America and by the Federal Ministry of Research and Technology of the Federal Republic of Germany. References i
W. Dejonckheere, W. Steurbaut, R.H. Kips - Pestic. Monit. J. I0, 68 (1976)
2
P.E. Corneliussen,
3
W. K8gel, W.F. MUller, F. Coulston, F. Korte - Chemosphere in press
- Pestic. Monit. J. 2, 140 (1969)
4
J.J. Betts, S.P. James, W.V. Thorpe - Biochem. J. 61, 611 (1955)
5
E.J. Kuchar, F.O. Geenty, W.P. Griffith, R.J. Thomas - J. Agr. Food Chem.
6
17, 1237 (1969) J.F. Borzelleca, P.S. Larson, E.M. Crawford, G.R. Hennigar Jr., E.J. Kuchar, H.H. Klein - Toxicol. Appl. Pharmacol.
7
M. Volz - priv. communication (Received in Germany 28 December 1978)
18, 522 (1971)