Studies on BHC isomers and related compounds

PESTICIDE

PHYSIOLOQY

4, 22e-231

Studies

on BHC

Isomers

Urinary

Metabolites

BIOCHEMISTRY

VIII.

AND

NORIO Radioisotope

Received

Produced Chlorophenol

KURIHARA

Research

and

AND

Related

from y- and ,&BHC Conjugates MINORU

Center and Department Iiyoto University, Kyoto,

September

24, 1973;

accepted

220 Copyright All rights

@ 1974 by Academic Press. Inc. of reproduction in any form reserved.

in the Mouse:

NAE~AJIMA of Agricultural Japan January

Metabolites produced from r-BHC and &BHC in the mouse characterized. Most metabolites from the both isomers were not solvents, and were conjugates such as sulfates and glucuronides. an appropriate enzyme, the conjugates gave chlorophenols, chlorophenol existed most abundantly-about 25% in the total Dichlorophenol constituted also a significant portion.

A number of metabolism studies have been performed on BHC (isomers of benzene hexachloride, or hexachlorocyclohexane, HCH) in insects, in microorganisms, in plants, and in mammals; especially on the y-isomer (lindane). Although mrtabolism studies in mammals in viva seem very important for the evaluation of their effects on man, there have been a relatively small number of this kind of studies. Grover and Sims (1) reported the prcsence of 2,3,5- and 2,4,5-trichlorophenol, their conjugates, and 2,4-dichlorophenylmercapturic acid in the rat urine after administered with the y-isomer of BHC intraperitoneally. Chadwick and Frcal (2, 3) proved the presence of dichlorophenols, trichlorophenols, tctrachlorophcnols, two isomers of tetrachlorocyclohexcnol, and 2,3,4,5,6-pcntachlorocyclohcx-2-an-l-01 as the metabolitcs in the rat urine of orally administered y-BHC. However, these two papers did not describe the abundance of the each metabolite. Karapally and coworkers showed in their recent two papers (4, 5) the existence and

Compounds

Chemistry,

4, 1974 urine were purified and extractable by organic After hydrolysis with among which 2,4,6-triurine metabolites. 2,4-

the abundance of several chlorophenols of the wide variety as well as ortho-dichlorobcnzcne as the metabolitcs of y-BHC in the rabbit. According to their reports, 2,3,5-, 2,4,5-, and 2,4,6-trichlorophenol were the principal metabolites. Freal and Chadwick (6) also reported recently the abundance of the each metabolite which they had idrntificd. Most papers dealt with only organicsolvent extractable portions of the metabolites ; thcreforc we have very little informations on the water soluble metabolitrs, among which several kinds of conjugates seem most important as suggested by the Grover’s report (1). Several years ago, WCreported the wholebody autoradiographic, study on the distribution of injected BHC isomers in the mouse. In connection with this study, the metabolism study was planned, and mainly water soluble metabolites in the urine havcl been examined. Although studies on t,hc y-isomer was performed most precisely, t)hc mctabolites from the B-isomer, which is most persistent in animal tissues (7, S) and

METABOLTTES

OF

BHC

ISOMERS

has highest chronic toxicity to mammals (Y), were also rxamincd. Some preliminary studies on the cu-isomer wtw also performed.

MATERIALS

ANI’J

: CHLOROPHENOL

221

CONJUGATES

TABLE

1

Thin T,ayer Chro~~~ogruph~c Data oj Possible Mdabelites Showing Relative Mobility to 2,4,5-Trichloropkrnyl-p-Dglucuronide

METHODS

Solventa

Administered

ChenGca1.s

Uniformly lab&d 14C-BHC isomers were prepared by Radiochrmical Center, UK, and purchased via Nippon Isotope Kyokai. They were thin layer chromatographically pure (over 987,). Th(l specific activities were: 155 &i/mg for y-isomer, 124 &i/mg for P-isomer, and 166 pCi/mg for cr-isomer. Cold r-BHC was obtained by photochlorination of bcnzcnc and purified by partition chromatography (tl-hrxane saturated with nitromcthanc as a mobile phase, and silicic acid mixcld with ,rL-hcxanc/ nitromethane as a stat,ionary phase) and recrystallization from ethanol to afford the gas chromatographic pure samples. An &ctron capture detector (“3Ni) was uwd as the d&&or in the gas chromatography. Jlclting point of pure y-BHC was 11YC.

Refercncc compounds such as mono-, di-, tri-, and tctrachlorophcnols wcw purchased and purified by recrystallizations and column chromatography. fl-n-Glucuronidcs of 2,4,5trichlorophenol and of 2,4,6-trichlorophcnol were synthesized by KijnigsKnorr condensation of the corresponding trichlorophenol with methyl (tri-O-acetyla-D-glUCOpyranOSy1 bromide)-uronatc (10). Methyl tri-O-acetyl-2,4,&trichlorophenyl+ n-glucopyranosiduronate of mp 173°C (Analytical value: C 44.52, H 3.907,) [Reported mp 165°C (l)], and its 2,4,6-isomer of mp 1725°C (Analyt’ical value: C 44.65, H 3.65, Cl 20.607,) (unreported) were obtained. Calculated analytical values for C~H&130~,, are: C 44.40, H 3.70, Cl 20.747& Alkaline hydrolysis (0.1 N-NaOH, 7O”C, 5 hr) of these crystalline condensates afforded the corresponding free glucuro-

Compound 2,4,5-trichlorophenyl-p-oglucuronide* 2,4,6-trichlorophenyl-fl-nglucuronide 2,4-dichlorophenylmercapturic acid S-2,4-dichlorophenyl-rysteine * standard

(I?, value

L1See the text

for

: the composition

(A)

(B)

CC)

1.00

1.00

1.00

1.02

1.03

1.00

1.03 1.00

1.05 0.75

0.88 0.87

0.42

0.71

0.83)

of the solvent.

nides. Each of them showed one spot on thin layer chromatograms in several solvent systems (Table l), and seemedthe expected glucuronide, although they were syrup. S-3,4-Dichlorophenyl-cystcinc and 2,4-dichlorophenylmercapturic acid were prcpared as described in the literature (11). The former decomposed at 1Sl”C (lit. lSO’C), and the latter melted at 159°C (lit. 160°C) and showed the following analytical values: C 42.59, H 3.64%. (Calculated values: C 42.86 H 3.579;; for C11H1103NSC13).

Enxymes

as Reagents

/3-Glucuronidase (bacterial, powder) and arylsulfatase (limpet, suspension in ammonium sulfate : fi-glucuronidase activity < 2% in relation to sp act of the sulfatase), wcrc purchased from Sigma Ltd. and Boeringer Mannheim, respectively, and employed without further purification in the hydrolysis of conjugates. Glucuronidase was added to the substrates in 0.1 M AcOHAcONa buffer (pH 5.05) and reacted at 37°C overnight. Arylsulfatase action was c.onducted in 0.1 211AcOH-AcONa buffer (pH 5.65) at 25°C.

222

KURIHARA

AND

NAKAJIMA

TABLE Fractionation

2

and Characterization

Fractionation

Characterization

Urine

TLC

(A)

TLk

(B)

l-7

Enzyme I

TLC

(A)

__

co-TLC

(A, B, C)

__

co-TLC

(A, B, C)

I Enzime

Extraction with Ether or Benzene

I Extraction with Ether or Benzene

TLC

TLC(D)

co-TLC (D)

-r Diazomethane (Enzyme

Diazomethane : Arylsulfatase

-co-GC

__

co-GC

(D, E) and (a, b) (a, b)

or /3-n-Glucuronidase)

Administration, Sample Collection, and PuriJication in the Radioisotope Experiments Four PCi of 14C-BHC in 0.2 ml of Ringer’s solution was injected to each mouse intraperitoneally once (12). Male mice (ddY, 4 wk old, 18-20 g wt) were used: two for the (Y- and p-isomer, resp, and six for the y-isomer. The doses were 22, 32, and 21 (or 16) pg for CY-, p-, and y-isomer, resp. The mouse was placed in a metabolism cage, and the urine was collected daily. Preparative tic on 200 X 200 (mm) plates was done for the purification of the radioactive components in the urine. Extraction of the each component detected by autoradiography from the tic plates was done by scraping off the radioactive zone, vigorous shaking

the obtained powder for at least 1 min with 50yo aqueous ethanol (before enzyme action) or benzene (after enzyme hydrolysis), and centrifuging to precipitate the powder. The extraction procedure was repeated at least three times for each scraped-off fraction. Purification processes of the metabolites are summarized in Table 2. Characterization

and Identijkation

Chromatographic characterizations, that is, co-thin-layer-chromatography and cogas-chromatography with reference compounds were performed. Detection of the radioactive zone on thin layer plates was done by means of autoradiography with X-ray film (Sakura N type), and radio-

METABOLITES

OF

BHC

ISOMERS

chromatographic scanner equipped with a gas-flow type thin window (or windowless) GM tube. The instruments were Nuclear Chicago Actigraph III and Aloka JTC-203. Radiogaschromatographic detection was performed by a gas-flow type proportional counter set in the column oven, and the simultaneously injected reference compounds wcrc detected by thermal conductivity detector. The instrument was Yanaco G-80. Counting gas was propane which was mixed with the carrier gas (helium) just after the t,hcrmal conductivity detector. The radioactivity of the each purified fraction was determined by liquid scintillation counters. The instruments wcrc Nuclear Chicago Isocap 300, and Horiba liquid scintillation spcctromct’cr. Scintillation cocktail was a solution of 6 g of 2,5diphenvloxazolc, 275 mg of p-his-[2-(k methyl-5-phenyloxazolyl)]-bcnzenc, and II:! g of naphthalcnc in 1 liter of p-dioxane. Methylation by diazomethane was somet,imcs performed on phenol fractions in order to confirm t.he assignment furt.hcr by co-gas chromatography with the authentic chlorophenol methyl ethers. A dilute solution of the sample in ,n-hexane was mixed with the freshly prepared diazomethane in ether. After 5 min standing of the resulting solution at room temperature, the mcthylation was complet’e. Chr*omatographic

Cotlditions

Thin layer chromatographic separations and analyses were carried out on silica gel G (Merck) plates using the following solvent system : (A) (B) (C) (D) (E)

n-BuOH/n-PrOH/2 N ammonia (2:l:l v/v) n-BuOH/AcOH/Water (5 : 1: 4 v/v, organic phase) Pyridine/ n-BuOH/ Water (1: 1: 1) Benzene/EtOH (19 : 1 v/v) Chloroform (not dried).

Gas chromatographic analyses were pcrformed under the following conditions :

: CHLOROPHENOL

Daily

223

CONJUGATES

UGury

TABLE

3

Excretion

of Radiouctivitp Isomer

Day

a

B

Y

1 2 3

24

44

2

6 2 2

Total

37

10

57

11

‘I Average percentages in

the administered

11

2

dose are

shown.

(4

(b)

1.5yo Neopentylglycol succinatc polyester on Chromosorb W AW (SO-SO mesh), 2.25 m; 150°C for free phenols, and 130°C for methyl ethers. 37, Silicone OV-17 on Chromosorb W AW DMCS (SO-80 mesh), 1.5 m; 125°C for free phenols, and 110°C for methyl ethers.

The tubes were U-shaped, and of 3 mm inner diam (glass). The carrier gas was helium, the flow rate of which was 20-25 ml/min. with the Cold y-Isomer; Mass Fragme,ntographic Ide,ntification of a Metabolite

Eqe~iments

Twelve mice were each given 45 i-e of cold y-BHC in Ringer’s solution by an ip injection. The urine collected during first 24 hr after dosing was examined by a similar procedure as described above in the radioisotope experiments. In the tic purification, radioactive urine was also applied at the both side ends of the same plate as an unmixed guide of the cold metabolites. The arylsulfatc fraction was extracted and hydrolyzed by the sulfatase. Ether extract of the reaction mixture was concentrated and further purified by tic. The purified metabolite hydrolysate was submitted to a mass fragmentographic analysis (13) as a solution in n-hexane. 1Iass fragmentography is a very sensitive analytical t,echnique detecting the ions of the specified m/e

224

KURIHARA

1st FIG.

(b:i:i).

Day

AND

NAKAJIMA

2nd Day

3rd Day

I’LC autoradiogram of nwtabolites from r-BHC. Solvent: n-BuOH/n-PrOH/:! F: front, 0: origin; S: sulfates (Fraction 3). G: glucuronides (Fraction 1).

1.

values during the gas chromatographic separation and requiring only nanogram to picogram of the material when we USCthe m/e value of the base peak in the mass spectrum of the expected authentic sample. This is not a gas chromatography-mass spectrum technique itself, and dots not give us such comprehensive informations on fragmentation patterns as the lattc>r. However, its sensitivity and specificity make it a very powerful tool for the identification of the compound of an infinitesimal amount. Details of the experimental conditions

N

Ammonia

are described in the explanatory Fig. 4. RESULTS

AND

notes of

DISCUSSION

Diffcrenccs in tha cxcrction rates of radioactivity between the @-and y-isomers has bern reported (12). /?-BHC seemsmuch more slowly metabolized than the r-isomer judging from the cxcrction rates. The excretion rate of the a-isomer was intermediate between these two (Table 3). Thin layer chromatography and autoradiography (Fig. 1) of radioactive components origi-

METABOLITES

OF

BHC

ISOMERS

: CHLOROPHENOL

TABLE Benzene-Soluble

Part

solution

22,000

part

from

(pH

= 4) Benzene

Aqueous

dpm

670,000

0.1

dpm

NaOH

N

Aqueous

part

2000 dpm 00%)

part

C@‘=%)1°

-

1

Extract)

(X2

(benzene-insoluble)

(3.2%)

Benzene-soluble (neutral) part

y-BHC

AcOH

~

1: Benzene-soluble

225

4

of Metabolites Urine I -

Acidic

CONJUGATES

Acidic

__

1 A’ H&O*

solution

(pH

-

C0.3%1

= 2)

Benzene

Benzene-soluble (acidic) part 20,000

dpm

lW%l a The

numbers

in brackets

are percentages

of the total

natcd from y-BHC suggested that the composition of the metabolites was not very different from day to day. Before tht first chromatographic purification, benzene extraction was tried. Only 3 and 11% of the total radioactivity was extracted from the urine of the mouse given y- and P-isomer, respectively. Most radioactivity (over 90%) in the benzene solution was extractable by 0.1 N NaOH, and was

urine

radioactivity.

acidic (Table 4). It seemed the mixture of 2,4-dichlorophenol, 2,4,6-trichlorophenol, and probably 2,4,5-trichlorophenol by a radiogaschromatographic examination (Fig. 2). The examination on the metabolites from y-BHC is described first. The each purified fraction from the benzene insoluble part by the procedures illustrated in Table 2 and 6 was charac-

226

KURIHARA

AND

NAKAJIMA

FIG. 2. Kadioyaschronlatogra?~~ of benzene-soluhlc part in the gamma-BHC urinary mctabolites. chromatographic condition: (a) (xc text). Prak (A) 2,4-DCP, (B) ,0,4,6-TCP, (C) J,4,5-TCP? scissa: Time (1 section: 0.5 min). Ordinate: IZadiodctcctor response.

TABLE Chromatographic

Data

5

of Chlorophenols

and Their

Methyl

Chlorophenols Relative

* Standard

mobility or relative t,ime to 2,4,5-TCPb

Relative retention time to 2,4,5-TCP methyl ether

GC(a)

GC(b)

1.13 0.63 0.61 1.00 1.00 1.29 1.60 0.86 0.92 1.00 1.00 1.29 1.06

1.03 0.48 0.48 1.00 1.00 1.08 1.33 0.53 0.67 1.00 1.00 1.22 1.03

0.13 0.76 0.76 0.35 0.34 0.35 0.33 2.94 2.38 0.79 1.00 0.69 2.16

0.16 0.48 0.50 0.43 0.42 0.41 0.48 1.31 1.21 1.00 1.00 1.10 2.85

0.41

R,:

0.49

Rt:

Methyl

retention

TLC(E)

R,:

Ethersa Chlorophenol Ethers

TLC(D) O-Cl-P m-Cl-P p-Cl-P 2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,4-DCP 3,5-DCP 2,3,5-TCP 2,4,5-TCP* 2,4,6-TCP 2,3,4,6-TTCP

Gas Ab-

7.3 m

Rt:

6.1 m

GC (a)

GC (b)

0.19 0.17 0.17 0.56 0.43 0.41 0.22 0.42 0.30 0.56 1.00 0.36 0.94

0.13 0.10 0.11 0.43 0.34 0.33 0.18 0.32 0.23 0.91 1.00 0.36 1.18

Rt:

7.7 m Rt:

16.6 m

a Retention times of the standard compounds are expressed in minutes. See text for the composition of the solvent systems in tic and for the gas chromatographic conditions. * Abbreviations employed in the Tables and Figures: TLC = thin layer chromatograhy, GC = gas chromatography, Cl-P = monochlorophenol, DCP = dichlorophenol, TCP = trichlorophenol, TTCP = tetrachlorophenol.

METABOLITES

(3F

BHC

ISOMERS

: CHLOROPHENOL

CONJUGATES

227

FIG. 3. Examples of racliogaschromatograms of partially puriJied fractions (Column b). (A) From gamma-BHC; glucuronidase hydrolysate of fraction 1, ether-soluble and acidic (peaks of 2,4-DCP and J,4,6-TCP). (B) From beta-BHC; arylsulfatase hydrolysatr of fraction 1, ether-soluble and acidic (peak of 2,4,6-TCP). (C) From gamma-BHC; arylsulfatase hydrolysate of benzene-insoluble part of the urine (peaks of 2,4,6-TCP and 2,4-DCP; Z&DCP in this case might have come from its glucuronidc conjugate, because of contamination of glucuronidase in sulfatase.). Abscissa: Time (1 section: 0.6 vain); Ordinate; Detector response: upper signals by radiodetector and lower signals by thermal conductivity detector. These signals were recorded after one section shift in abscissa from each other.

tcrizcd by co-thin-layer-chromatography before and after the enzyme hydrolysis; co-gas-chromatography after the hydrolysis; and co-gas-chromatography with the methyl ethers of phenols after methylation. Figure 3 shows some examples of double pen recorded co-gas-chromatograms of the metabolite hydrolysates at partially purified stages. These characterization procedures distinguish clearly each chlorophenol from others (see Table 5). About 60% of the fraction 1 (having smaller R, values in TLC(A) : see Table 6) was hydrolyzed by fl-glucuronidase, among which 2,4,6-trichlorophenol and 2,4-dichlorophenol were principal phenol components.

Fraction 3 (of larger values in TLC(A) : see Table 6) consisted mostly of one component which afforded, after purification and arylsulfatase hydrolysis, 2,4,6trichlorophcnol almost exclusively, though there seemed to be present a small amount of 2,4-dichlorophenol. Comparisons of chromatographic data of the each metabolite fraction with those of the expected authentic samples are listed in Table 7. 2,4,6-Trichlorophenol from the sulfate fraction was further examined by mass fragmentography (Fig. 4). In this case, the ,m/e values detected were 196 and 198, which correspond to molecular ion (;\I+) (C~H3035C13)+and the ion (RI + 2)+ : (CeHa035C1237Cl)+(see Fig. 5). Rf

228

KURIHARA

AND

NAKAJIMA

TABLE Rcsdts

6

oJ Fractionation” Urine ~

Benzene I Benzene (2220%)

Aqueous W-90%‘,) ~

I

I

I

TLC

I

II

(A)

I

I

(42170) I-

TLC(B)

IF

TLC(B)

i-

TLC(B)

34

(3%)

(7%) ~

TLC

133

134

(65%)

(23%)

(A)

--

(90%) TLC

Glucuronidase

___

Aqueous

Ether

Ether 133E (90%) ~

(37%)

Arylsulfatase

~

Ether

IAqueous

~

Ether 34E @5f%‘o) __

~

Aqueous

TLC

~

254

CO.9%1 __

(A)

3433 (95%)

Ether 134E (95+%) (D,

__

TLC(D)

2,4,6-TCP TLC(D)

W%l

One component? 13332 (33%)

133E3 (33%)

2,4-DCP

2,4,6-TCP

Acidic

C7%1 C5%1

Cs%l

a Yields in each separation step activity are presented in brackets.

are shown in parentheses, while the percentages in the total urine The fractions studied further are numbered by Arabic numerals.

The metabolites from the p-isomer wcrc similarly examined. The metabolites thus characterized are summarized in Table 8. Principal metabolites from the both isomers were 2,4,6-tri-

radio-

chlorophenol conjugates-sulfates and glucuronides. They constituted about onefourth of the total urinary metabolites. The other identified phenol component was 2,4dichlorophenol, the conjugates of which

VETABOLITES

OF

BHC

ISOMERS

: CHLOROPHENOL

TABLE Chromatographic

7

Comparison of Fractionated Metabolites with Relative Mobility or Relative Ret&ion TLC

Fr. 133 Fr. 134* 2,4,6-TCP-Gl.* 2,4,5-TCP-GLc

(A)

Authentic Tiwle

TLC(B)

1.01 1.00 1.02 1.00

229

CONJUGATES

Specimens

Showing

TLC(C)

1.03 1.00 1.03 1.00

1.00 1.00 1.00 1.00 (Standard)

TLC(D) 1.00 1.29 1.00

TLC(E) 1.00 1.23 -

GC(a) 0.34 0.69 0.13

GC(b) 0.42 1.10 0.11

(Methyl GC(a) 0.43 0.36

ether) GC(b) 0.34 0.36 -

Fr. Fr. Fr.

13332 13333 134E”

Fr.

3433

1.28

1.22

0.60

1.10

0.36

0.36

2,4,6-TCP 2,4-DCP 2,4,5-TCP (St‘andard)

1.29 1.00 1.00

1.22 1.00 1.00

0.69 0.34 1.00

1.10 0.42 1.00

0.36 0.43 1.00

0.36 0.34 1.00

a Not identified with any chlorophenol b 2,4,6-Trichlorophenyl-B-n-glucuronide. c 2,4,5-Trichlorophenyl-@-n-glucuronide.

or conjugate

constit’utcd also a significant portion of the total metabolites from the both BHC isomers. Thcrc were several fractions which were partially characterized but not identified yet. Glucuronides with unidentified acidic volatile aglycon(s) (fraction 134E in Table 6) and those with neutral one(s) (in the fraction 133E in Table 6) wcrc found. The former seemed a phenol of high volatility, AUTHENTIC I

SOLVENT HEXANE I

SAMPLE I

FIG. 4. Massfragmentograrn of 2,4,6-TCP. (Authentic sample, solvent, and sample fraction corresponding lo fr.S.&W.) (Temperature: ion source 27O”C, ,molecular xparator 220°C; Electron energy: 20 eV; Trap current: 60pA; Ace. H. V. 35kV. GC coluwln 1.5% NPGS on Chrowlosorb W AW DMCS (60-80 mesh), 1 m, 180°C; Carrier gas: Helium SO ml/min; m/e for mass fragmentography: 196 and 198; Recorder speed: 10 mm/min.)

examined.

but was different from any chlorophenols examined. The latter(s) might be such polychlorocyclohexenol as described by Chadwick and Freal (2, 3). Some other types of water soluble mctabolites were also found. Fraction 2 in Table 6 gave a component (fraction 254) behaving like 2,4-dichlorophenylmercapturic acid on tic examinations, and affording an ethersoluble radioactive material quantitatively after heating in an alkaline solution (1 N ,oo?~

1% M’

1

co

40

60

80

FIQ. 5. Mass Spectrum Experimental conditions

x)0

120

WI

WI

93(MJP

I m/e co

of the Authentic I,4,6-TCP. are described under Fig. 4.

230

KURIHARA

AND

NAKAJIMA

TABLE Abundance

of the Principal

8

Urinary

Metabolites

Glucuronide From

Neutral

2,4,6-TCP L 2,4-DCP (not identified)a (not identified)b

Conjugates, Total Free Chlorophenok

Total 25-26 4-6 7-8 5-6

5-6 4-5 7 5-6

;“p, (1) -

21-24

20-22

41-46 3

15 l-2 +

10

25 4-5 +

16-18

13

p-BHC 2,4,6-TCP 2,4-DCP 2,4,5-TCP

Conjugates, Free, Acidic

Total (not identified)

a Fr. 134E (see Table 6). b In the fraction 133E (see Table 6). c 2,4,6-TCP> 2,4-DCP( > 2,4,5-TCP)

TABLE Reported

9

Metabolites

of y-BHC

Rabbitso-Dichlorobenzene 2,3-DCPd 2,4-DCPd 2,3,5-TCPd 2,4,5-TCPd 2,4,6-TCPd 2,3,4,6-TTCPd PCCOL” Others (identified) identified

3.4c 2.7 2.5 8.1 8.6 7.2 3.4

Rats”

+++ ++ ++ ++ +++

3.1 39.0

Q Ref. (4) and (5). b Ref. (6). =Percentages in the total urinary d Free chlorophenol. e 2,3,4,5,6-Pentachlorocyclohex-2-en-l-01.

3 -

29-30 11

(see text).

NaOH, lOO”C, 3 hr) just like 2,4-dichlorophenylmercapturic acid affording 2,4-dichlorothiophenol. However, tic examinations of the ether-soluble alkaline hydrolysate from the fraction 254 showed its incon-

Total

Sulfate

r-BHC

Acidic

From

in the Mouse

metabolites.

sistcncy with 2,4-dichlorothiophenol (and its oxidized form). This fraction seems to include certain mercapturic acid derivative(s), but it was not further studied because of the small amount available (less than 1% of the total urine metabolites). The most significant characteristics of the present results are the abundant existence of the conjugates of chlorophenols instead of their free forms, and an unusually high percentage of 2,4,6-trichlorophenol and 2,4-dichlorophenol among the chlorophcnol components. Any mercapturic acids were not identified yet and they seemed to bc present in a very small amount, if any. Several metabolites from r-BHC have been reported as summarized in Table 9, which shows the abundant existence of free chlorophenols of wide variety. A report described the presence of 3,4-dichlorophenylmercapturic acid as a metabolite of r-BHC. For the &isomer, a report dcscribed free 2,4,6-trichlorophenol as a sole identified metabolite in the rat.

METABOLITES

OF

BHC

ISOMERS

These interesting differences between the present results and others may be due to the speciality of the mouse, and also due to the administration procedure-sinyle intraperitojleal injection in the present case, instead of continuous feeding or daily injections during a period of several days in the other cases. According to a recent private communication to the present authors (3), Real found an appreciable amount of chlorophenols cxist,ing as their conjugates. Also Karapally and coworkers (5) described the presence of the ether insoluble portion (44% of the total), which can bc chlorophenol conjugates. In both cases, however, free chlorophenols were present as abundantly as their conjugates: different from our present, results. ACKNOWLEDGMENTS

We are grateful to Dr. Hideyo Shindo and Dr. Eiichi Nakajima of Sankyo Co. for their help in the animal experiments; to Mr. Shuhei Nakajima of Dept. of Agricultural Chemistry, this University, for his preliminary purification of the metabolites; and to Mr. Takeshi Murata and Mr. Tsunezo Takeda of Shimadzu Seisakusho Co. for t,he mass fragmentographic analysis. REFERENCES

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