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)