Covalent binding of [14C]methoxychlor metabolite(s) to rat liver microsomal components

TOXICOLOGY AND APPLIED PHARMACOLOGY68, 367-374 (1983)

Covalent Binding of [‘4C]Methoxychlor Metabolite(s) to Rat Liver Microsomal Components’ WILLIAM

H. BULGER, JANE E. TEMPLE, AND DAVID KUPFER~

Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545

Received August 14. 1982; accepted January 13, 1983 Covalent Binding of [‘%]Methoxychlor Metabolite(s) to Rat Liver Microsomal Components, BULGER, W. H., TEMPLE, J. E., AND KUPFER, D. (1983). Toxicol. Appl. Pharmacol. 68, 367374. [“ClMethoxychlor was incubated with NADPH-fortified liver microsomes from male rats, and covalent binding to microsomal components was determined. The binding process was markedly enhanced when microsomes From phenobarbital-treated rats were employed. However, when microsomes from methylcholanthrene-treated rats were used the level of binding was not significantly al&&d. Incubation in the presence of ghrtathione, cysteine, or ascorbate markedly diminished binding Metyrapone and SKF 525-A, inhibitors of hepatic cytochrome P-450-linked monooxygenase activity, inhibited the binding. Also, ethylmorphine and hexobarbital, alternate substrates of the monooxygenase system, inhibited binding There was no binding to microsomal components in the absence of NADPH or oxygen. TCPO (1, I, I-trichloropropane-2,3-oxide), an inhibitor of epoxide hydrase activity, failed to enhance the binding process. However, N,N’diphenyl-p-phenylenediamine (NDP) and n-propyl gallate (PG), both free radical scavengers, decmased binding at micromolar concentrations without altering the extent of formation of polar [“C]methoxychlor metabolites. It was concluded that methoxychlor undergoes a hepatic microsomal monooxygenase(s)-mediated activation and that the resultant reactive metabolites (possibly free radicals) bind covalently to microsomal components. By contrast, the binding resulting from the incubation of an impure mixture of polar [“C]methoxychlor metabolites with liver microsomes did not require NADPH and O* and was not affected by NDP, PC, ascorbate, or heat-treatment of microsomes. This finding suggested that the binding subsequent to the initial metabolic activation of methoxychlor does not require further enzymatic transformation. However, whether the binding with metabolites represents the same chemical species as the binding with [‘4C]methoxychlor remains to be established.

Methoxychlolj is a structural analog of DDT with insecticidal properties. In recent years methoxychlor has enjoyed increasing usage as ’ This work was presented in part at the 64th meeting of the Federation of American Societies for Experimental Biology, April 15-23, 1982, New Orleans, Louisiana (Fed. Pm. 41(5), abstract 7620). * To whom correspondence should be addressed. 3 Abbreviations and common names of DDT analogs: 1, 1, I-trichloro-2,2-bis(p-methoxyphenyl)ethane, methoxychlor, 1, 1,l -trichloro-2,2-bis(pchlorophenyl)ethane, 1,l dichloro-2,2-bis(p-chlorophenyl)ethane, P.PDTT; l,l,l -trichloro-2,2-bis-(p-hydroxyphenyl)P.P’DDD; ethane, HPTE. 367

a substitute for DDT. Its main advantages over DDT reside in a low acute toxicity in mammals and in a diminished persistence in the biosphere (Murphy, 1980). Numerous xenobiotic compounds undergo metabolic activation resulting in the formation of chemically reactive intermediates, which may blind covalently to cellular macromolecules. Often, the activation step is mediated by the hepatic cytochrome P-450-linked monooxygenase system(s). In many casesthese interactions have been implicated as causative factors in numerous toxic responses (Brodie et al., 1971; Gillette et al., 1974; Reid 0041-008X/83

$3.00

Copyright 0 1983 by Academic Press. Inc. All rights of i-epmduction in any form resewed

368

BULGER,

TEMPLE.

and Krishna, 1973; Gillette, 1974a,b) including carcinogenesis (Miller and Miller, 1966; Miller, 1970; Weisburger and Weisburger, 1973; Gillette et al., 1974). The impetus for this investigation is based on two factors: (a) methoxychlor was reported to be carcinogenic4 to the liver as well as to other organs in laboratory animals (Reuber, 1980), and (b) during metabolism studies with [‘4C]methoxychlor and NADPH-fortified rat liver microsomes, we observed that in addition to the formation of phenolic metabolites some of the radioactivity was strongly associated with the microsomes. It appeared reasonable that a reactive metabolite may be involved in methoxychlor toxicity. This investigation was initiated as the first step in exploring this possibility and attempts to accomplish the following: (a) establish the in vitro covalent binding,’ (b) demonstrate involvement of the cytochrome P-450-linked monooxygenase in the binding process, and (c) examine the nature of the reactive intermediate(s) involved in the covalent binding. METHODS n-Propyl gallate, cysteine, glutathione, EDTA disodium salt, NADPH, glucose-6-phosphate, and glucose-6phosphate dehydrogenase were from Sigma Chemical Co. (St. Louis, MO.). N,N’-Diphenyl-p-phenylenediamine and 3-methylcholanthrene were from Eastman Kodak (Rochester, N.Y.). I, I, I -Trichloropropane-2,3-oxide was from Aldrich (Milwaukee, Wise.). Ascorbic acid was from ICN Pharmaceuticals (Cleveland, Ohio). Ethylmorphine. HCI was from Merck (Rahway, N.J.). Phenobarbital sodium was from Mallinckrodt (St. Louis, MO.). Corn oil (USP grade) was from Matheson Coleman and Bell (Cincinnati, Ohio). Compressed gases were from Linde. SKF 525 A. HCl was a gift from Smith Kline and French Laboratories (Philadelphia, Penn.). Metyrapone, also a gift.

4 Whether this activity was due to genotoxic action or via an epigenetic mechanism has not been established; however, with respect to carcinogenesis of DDT, epigenetic mechanisms have been proposed (Williams, 1981). ’ In this manuscript, although not proven rigorously, covalent binding is assumed when radioactivity remains associated with microsomal components after extensive extraction with a variety of organic solvents.

AND

KUPFER

was provided by Ciba Pharmaceutical Co. (Ardsley. N.Y.). Ins&Gel was from Packard (Downers Grove, Ill.). Liquifluor was from New England Nuclear (Boston, Mass.). Uniformly ring-labeled [“Qmethoxychlor (1.8 mCi/ mmol) was obtained from California Bionuclear Corporation (Sun Valley, Calif.). Routinely, the [14C]methoxychlor was purified by a previously described extraction method (Bulger et al., 1978). The radiochemical purity of 99+% was established by tic employing solvent systems 1 to 3 (see below). Male Sprague-Dawley CD rats (110 to 120 g) were obtained from Charles River Breeding Laboratories and were treated with phenobarbital or 3-methylcholanthrene. Phenobarbital treatment (37.5 mg/kg ip in 0.2 ml Hz0 twice daily) was for 4 days, and liver microsomes were prepared 12 hr after the last injection. Rats treated with 3-methylcholanthrene (25 mg/kg, ip, in 0.4 ml corn oil) were injected once daily for 3 days, and liver microsomes were prepared 48 hr after the last injection. Control animals for each treatment group were injected with vehicle only. The preparation of liver microsomes was as previously described (Burstein and Kupfer, 197 1). Incubations were carried out in 20-m] glass scintillation vials containing the following constituents: 0.6 ml (60 rmol) of sodium phosphate buffer (pH 7.4); 0.1 ml (10 pmol) MgC&; microsomal suspension ( 1 to 1.5 mg protein in 0.1 ml 1.15% aqueous KC]); EDTA (1 rmol); [‘4C]methoxychlor (100,000 dpm in 5 ~1 ethanol) or polar metabolites of [‘*C]methoxychlor (20,000 dpm in 5 ~1 ethanol); NADPH-regenerating system (glucose-6-phosphate, 10 rmol; NADPH, 0.5 pmol; glucose-6-phosphate dehydrogenase, 2 IU) in 0.1 ml (10 rmol) phosphate buffer and Hz0 to render a final volume of I ml. The reaction was initiated by adding the regenerating system and incubating at 37°C in a water-bath shaker. Except where indicated, incubations were conducted under an atmosphere of air. The reaction was terminated by adding 10 ml of ethanol. During the initial studies, the incubation mixture after ethanol addition was transferred to a 20-ml extraction tube. The content was mixed vigorously with a Vortex Geni mixer for 2 min and centrifuged at SOOg for 10 min; the supematant fraction containing unbound radioactivity was discarded. The resulting pellet was subjected to repeated washings by the above procedure employing 10 ml of the following solvents: ethanol. one time; hexane, four times; methanol-ether (3: l), six times; methanolether (1:3), four times. The final pellet was solubilized with 1 ml of 1 N NaOH at 60°C for 1 hr. The radioactive content of a 0.8-ml aliquot was determined by liquid scintillation counting employing 10 ml of Insta-Gel and 2 ml Hz0 in a Packard Tri-Carb 460 CD spectrometer. However, to facilitate the extraction procedure for a large number of incubations, the following modification was developed. After termination of the incubation with 10 ml of ethanol, the precipitate was trapped on a 2.4-cm Whatman GF/C glass microfiber filter adapted to a vac-

COVALENT

BINDING

OF [‘4C]METHOXYCHLOR

uum filter flask with a Schleicher and Schuell filter holder. The trapped precipitate was washed by passing the following solvents through the filter: ethanol, 10 ml; hexane, 40 ml; methanol-ether (3:1), 60 ml; and methanol-ether (1:3), 40 ml. At this point there was little or no radioactivity in the final wash. The filter was placed at the bottom of a 20-ml glass scintillation vial containing 2 ml Liquifluor, and the radioactive content was determined. Results obtained with the filter method were congruent with those obtained by the above more laborious procedure. Therefore, the filter procedure was employed throughout this study. Analysis by SDS-PAGE of trapped precipitates eluted from the filters and subsequent autoradiography revealed radioactivity in Coomassie brilliant blue-stained regions of the gel. However, no radioactivity was associated with proteins from incubations carried out in the absence of NADPH or when [‘%]methoxychlor was added during the SDS-mediated denaturation process. This finding provides further evidence that the radioactivity associated with the precipitate on the filter represents covalent binding to proteins. Polar metabolites of [%]methoxychlor were determined in the following manner. The ethanol fraction (20 ml) from the above filtration procedure was saved and evaporated to dryness with a stream of nitrogen. The resulting residue was dissolved by adding 3 ml of 0.67 N NaOH. Unmetabolized [Wlmethoxychlor was removed by extraction with hexane (four times with 6 ml each), and the resulting aqueous phase was acidified (pH I to 2) with 1 ml 2 N HCI. The radioactive content of 2 ml of the acidified fraction was determined by liquid scintillation counting employing 10 ml Insta-Gel containing 2 ml of water. Polar metabolites for incubation with microsomes were obtained by extracting the above acidic phase with ether (three times with 4 ml each). The ether fractions were pooled, evaporated to dryness with a stream of nitrogen, and taken up in an appropriate volume of ethanol to yield ca. 20,000 dpm/5 ~1. Protein determination was by the method of Lowry et al. ( 195 I ) with the modifications of Stauffer ( 1975). Thinlayer chromatography (TLC) was carried out with precoated, 5 X 20 cm, Silica gel 60, plates (EM Reagents) with a layer thickness of 0.25 mm. Plates were activated at 100°C for 1 hr in a vacuum oven prior to use. Four solvent systems were employed: (1) ether-hexane (3:l); (2) petroleum ether-chloroform-methanol (3:2: I); (3) hexane-acetone (4: 1); and (4) hexane-acetone (1: I). Alter chromatography, localization of radioactivity was achieved by scraping 1 cm zones of the gel into vials containing 1 ml methanol and 5 ml Liquifluor (Bulger et al., 1978).

RESULTS Preliminary experiments revealed that [‘4C]methoxychlor incubated with NADPH and liver microsomes from phenobarbital-

369

treated rats resulted in the incorporation of radioactivity into microsomal components, whereas incubation in the absence of NADPH did not. Figure 1 depicts the incorporation of radioactivity into microsomal components with respect to time. When microsomes from phenobarbital-treated rats were employed, the effect was dramatic. There was a marked (with respect to control values) increase in binding which remained linear for approximately 10 min. Thereafter the rise continued in a nonlinear fashion. Although it was not as overt, binding also occurred when control microsomes (from vehicle-treated rats) or microsomes from methylcholanthrene-treated animals were employed. Incorporation of radioactivity occurred approximately 10 min after incubation and remained linear for the subsequent 20 min. The lag period which occurred within the first 10 min of incubation remains unexplained; this lag may be due to a requirement for generation of a critical concentration of activated metabolites. In all subsequent experiments, a IO-min incubation time was employed with microsomes from phenobarbital-treated rats, and an incubation time of 30 min was used with microsomes from vehicle- and methylcholanthrene-treated animals. The study represented by Table 1 employed liver microsomes prepared from individual animals to demonstrate the effect of phenobarbital and methylcholanthrene treatment on the binding process of [14C]methoxychlor metabolites to microsomal components. Phenobarbital treatment increased binding by approximately lo-fold. Covalent binding to microsomes from methylcholanthrene-treated rats when compared to the appropriate control was somewhat diminished (Fig. 1 and Table 1). However, as indicated in Table 1, the difference was not significant. Also, as expected, cytochrome P-450 and cytochrome P-448 values were elevated in microsomal preparations from phenobarbitaland methylcholanthrene-treated rats, respectively. Table 2 represents the in vitro effect of various compounds on the microsomal bind-

370

BULGER,

TEMPLE,

AND KUPFER

7-

6-

TIME (min.) FIG. I. Time course of in vitro covalent binding of [‘4C]methoxychlor metabolite(s) to liver microsomes from phenobarbital- and metbylcholanthrene-treated male rata. Phenobarbital (O), control for phenobarbital (0), methylcholanthrene (A), control for methylcholanthrene (A). TABLE 1 COVALENT BINDING OF [%]METHOXYCHL~R METAROLITE(S) TO LIVER MICRO~OMES PREPARED FROM RATS TREATED WITH PHENOBARBITAL 0~ ~-METHYLCHOLANTHRENE'

Treatment Phenob%rbital Control(H#) %Methylcholanthrene Control (corn oil)

Binding (“C]methoxychlor equivalents (pmol/min/mg protein) 271.4 26.8 25.6 31.3

j, 20.7': k 5.3 + 3.5* % 2.0

Cyiochmme P-450(44.S)~

(nmollmg prwzin) 1.54 0.61 1.16 0.60

‘Binding data represent x‘ -C SE of values obtained with micmsomcs pm pared from individual rats (four rets per group). Statistical analysis by Studears f tea 'P-450 (448) values M fmm a si& determination employing pooled miaosome rmmred from the same animals wd in the binding study. O.OS,(NS), when coqwed to appnqiate cootrd.

ing of [‘4C]methoxychlor metabolite(s). Glutathione and cysteine at a 1 mM concentration inhibited binding to 68 and 27% of control, respectively. The inhibitors of cytochrome P-450 monooxygenase activity, SKF 525-A (0.5 mM) and metyrapone (1 mM), diminished binding to 4 and 5% of control, respectively, and saturating levels of an alternate substrate for microsomal monooxygenase, ethylmorphine (8 mM), decreased binding to 23%. Hexobarbital, another alternate substrate, also diminished covalent binding to liver microsomes (data not depicted). As indicated in Table 2, the fortification of the incubation medium with oxygen enhanced binding (with respect to an air atmosphere), whereas exclusion of oxygen (nitrogen envi-

COVALENT

BINDING

TABLE 2 Emcr

OF SULFHYDRYL

COMPOUNDS,

INHIBITORS

OF

MONOOXYGENASES, ALTERNATE SUBSTRATE, AND MOSPHERIC CONTENT ON COVALENT BINDING

ATOF

[‘%]METHOXYCHLOR CROSOMES PREPARED BATS

MI-

OR METABOLITE(S) TO LIVER FROM PHENOBARBITAL-TREATED

Experiment

Cysteine, Cysteine,

1 mM 5 mM

1 mM 10 mM

SKF525A,b Metyrapone, Ethylmorphine, Minus NADPH

0.5 mM 1 mM 8 mM

OxygenC NitrogenC

483.7 328.6 229.7 --21.5 22.5

of atmosphere

(100)” (68) (47)

193.8 -

(4) (5)

112.5 (23) 9.1 (2) ---

0 Values in parentheses represent ’ &Diethylaminoethyl-2,2diphenylpentanoate. ’ Instead

Experiment

2

Binding [‘4C]methoxychlor equivalents (pmol/ min/mg protein)

Additions/Omissions None (control) Glutathione, Glutathione,

1

(100)’ -

51.4 35.0

(27) (18)

-

-

1.1 (cl) 243.8 (126) 5.4 (3) percentage

371

OF [“CIMETHOXYCHLOR

of control.

of air.

ronment) markedly diminished binding. Addition of the epoxide hydrase inhibitor, 1, l,ltrichloropropane-2,3-oxide (TCPO), had little or no effect on the binding process at concentrations as high as 0.2 mM. However, higher concentrations of TCPO inhibited binding (Table 3, Experiments 1,2, 3). Clearly there was no potentiation of binding over the concentration range of TWO employed. As demonstrated in Table 4 (Experiments 1 to 3), micromolar concentrations of the free radical scavengers (NjV’diphenyl-pphenylenediamine (NDP) and n-propyl gallate diminished the binding process. Binding was also inhibited by 0.5 m&t ascorbate (Experiment 3). Of the three compounds, NDP appeared to be the most potent inhibitor. The inhibition of covalent binding was not accompanied by a decrease in the formation of polar metabolites (Table 4, Experiment 3), indicating that the inhibition of binding was not a reflection of diminished metabolite for-

mation. The exception was the high concentration of propyl gallate (1 mM), which inhibited both binding and metabolite formation (Experiment 3). Incorporation of radioactivity into microsomal components was observed when incubation was carried out employing polar metabolites of [‘“Clmethoxychlor (described below) in place of [‘4C]methoxychlor (data not depicted). The incorporation of radioactivity, which was substantially lower as compared with methoxychlor, increased almost linearly up to 10 min. The binding did not require NADPH, was not inhibited by propyl gallate (100 PM), NDP (1 PM), or ascorbate (0.5 mM), did not require oxygen, and presumably was not enzymatic (heat treatment of microsomes did not diminish incorporation of radioactivity into microsomes). Analysis of the polar metabolites of [‘4C]methoxychlor by TLC employing solvent system 4 (see Methods) revealed two regions of radioactivity. One (Rr 0.54) which comigrated with HPTE, the didemethylated metabolite of methoxychlor (Kapoor et al., 1970; Bulger et al., 1978; Ousterhout et al., TABLE

3

EFFECT OF THE EPOXIDE TRCHLOROPROPANE-2,3-OXIDE

HYDRASE INHIBITOR l,l, l(TCPO) ON COVALENT

BINDING OF [WIMETHOXYCHLOR LIVER MICR~SOMES PREPARED TREATED

FROM

METABOLITE(S) PHENOBARBITAL-

TO

RATS Experiment

Concentration

of

1

Experiment

2

Experiment

Binding [ Wlmethoxychlor equivalents (pmol/min/mg

TCPO

protein)

None

266.3

451.8

357.9

0.001, 0.10,

mM mhi 0.20,mM

290.2 274.4 -

478.2 457.5 -

348.6 347.6

0.50,mM 0.75,mM

161.1 -

223.3 -

1.00,

mM

1.50, 2.00, 5.00, None,

mM rnM mM minus

221.9 -

327.9 -

319.1 286.6 282.6

NADPH

104.7 6.7

131.2 3.0

249.3 212.9 2.7

3

372

BULGER, TEMPLE,

AND KUPFER

TABLE 4 EFFECX OF FREE RADICAL SCAVENGERSON THE FORMATION OF POLAR METALQLITES OF [14C]M~~~~~~~~~~ AND ON THE COVALENT BINDING OF [14C]M~~~~~~~~~~~ METABOLITE(S) TO LIVER MICROSOMESPREPAREDFROM PHENOBARBITAL-TREATED RATS Experiment 1

Additions/omissions None NDP’ NDP NDP NDP

(control) 0.1 PM 1.0 /.kM 10.0 PM 100.0 PM

None (control) PGd 1 jtM PG 10 PM PG 100 pM F’ci 1000 I.LM None (control) Ascorbate 0.5 mM None, minus NADPH

Experiment 3

Experiment 2

Metabolites” (nmol/lO min/mg protein)

Binding [‘%Z]methoxychlor equivalents (pmol/min/mg protein) 218.5 (1OO)b 152.7 (70) 139.5 (64) 46.0 (21) --

282.0 254.9 194.0 98.1 20.8

( 100)b (90) (69) (35) (7)

254.5 (100) -129.8 (5 1) 78.5 (31) 12.5 (5)

309.1 (100) 333.3 (108) 244.2 (79) 116.6 (38) 11.6 (4)

--2.3 -

--3.5 -

334.3 340.6 211.1 108.5 --

(1OO)b (102) (63) (32)

20.9 21.1 22.4 20.1 --

(lOO)b (101) (107) (96)

389.4 (100) -242.6 (62) 119.8 (31) 10.6 (3)

23.1 -24.9 25.0 8.8

(100)

355.0 (100) 110.7 (31) 1.4 -

21.7 (100) 24.2 (112) 0 -

(108) (108) (38)

’ Polar metabolites were determined in Experiment 3 only (see Methods). ’ Values in parentheses represent percentage of control. ’ N,N’-Diphenyl-pphenylenediamine (NDP), added to incubation mixture in 10 ~1 acetone. Control received 10 pl acetone. d n-Propyl gallate (PC), added in 0.1 ml HzO.

1981),6 contained 29% of the radioactivity. The remainder of the radioactivity, probably multihydroxy products, was found at the origin of the chromatogram. No radioactivity was associated with methoxychlor (Rf 0.77). DISCUSSION * Based on the above results it was concluded that methoxychlor is activated by a cytochrome P-450~linked monooxygenase system(s) and that the resulting reactive inter6 We assume that this radioactive metabolite is in fact HPTE. We have previously identified HPTE as a metabolite from incubations of methoxychlor with rat liver microsomes using HPLC and CC/MS procedures (unpublished).

mediate(s) binds in a covalent manner to hepatic microsomal components. The following summarizes the evidence for the involvement of cytochrome P-450~linked monooxygenase(s): (a) the binding process is dependent upon NADPH and oxygen, (b) liver microsomes prepared from animals treated with phenobarbital, an inducer of cytochrome P450-linked monooxygenase activity, markedly enhanced the binding process, (c) SKF 525-A and metyrapone, known inhibitors of cytochrome P-450-linked monooxygenase activity (Anders, 1971), diminish binding, and (d) alternate substrates for the monooxygenase system, such as ethylmorphine and hexobarbital, inhibit the binding process. Inhibition of the binding process by glutathione and cysteine suggests the formation of a reactive epoxide (Brodie et al., 1971) or a free radical

COVALENT

BINDING

OF [‘%]METHOXYCHLOR

(Misra, 1974; Goyal and Armstrong, 1975; Schaich and Karel, 1976). However, the failure of TCPO, an epoxide hydrase inhibitor (Oesch et al., 1971), to enhance the binding process suggests that the reactive intermediate is not an epoxide. Inhibition of the binding by TCPO at concentrations of 0.5 to 5.0 InM (Table 3) probably reflects the action of TCPO as an inhibitor of cytochrome P-450~linked monooxygenase activity (Yang and Strickhart, 1975; Prough et al., 1976; Shimada and Sato, 1979; Ivanetich et al., 1982). This observation is consistent with the report (Shima& and Sato, 1979) that TCPO inhibits especially species of cytochrome P-450 which are inducible by phenobarbital. The inhibition by TCPO of the binding process also sup ports our conclusion that the binding involves cytochrome P-450-linked monooxygenase. Also, the observation that the binding process was enhanced by microsomes from phenobarbital-treated animals but not by microsomes from methylcholanthrene-treated rats (Fig. 1 and Table 1) indicates that the activation step involves a specific class of monooxygenase(s). The diminished binding caused by micromolar amounts of the free radical scavengers NDP and propyl gallate (Slater and Sawyer, 197 1; Uehleke et al., 1973) suggests that the reactive intermediate(s) responsible for the binding process is a free radical(s). If this is the case, then it is tempting to postulate that the free radical(s) is formed by the loss of one or more chlorine atoms from the C, of the ethane unit of methoxychlor. However, other possibilities exist (Nelson, 1982), for instance, the active intermediate may be a demethylated-hydroxylated metabolite (catechol) which assumes a quinone-like structure. Such a compound should be capable of reacting with glutathione, cysteine, and microsomal proteins. Clearly it will be necessary to isolate and characterize the intermediate(s), free or bound, before this aspect of the binding process is understood. Preliminary experiments involving binding of isolated impure metabolites of [‘4C]methoxychlor demonstrated the presence of a relatively stable “ac-

373

tivated product,” which survived an extensive isolation procedure, and which does not require further metabolic transformation for initiating binding activity (see Results). The observation that the incorporation of radioactivity from the isolated metabolites is not significantly inhibited by NDP, propyl gallate, and ascorbate, whereas these compounds were strongly inhibitory when [r4C]methoxychlor was employed, suggests that the activated intermediates are not the same in the two cases. Our results with anaerobic conditions are interesting when contrasted with a study employing p,p’DDT. In a brief communication, Baker et al. (1982) showed that anaerobic conditions enhanced p,p’DDT binding to rat liver microsomal components whereas in this study (Table 2) the opposite was observed. Although methoxychlor and p,p’DDT are structural analogs, they apparently are activated to bind to microsomal components by different mechanisms. Interestingly, the binding of a metabolite of p,pDDT, p,p’DDD, to microsomal components requires an atmosphere of oxygen (Baker et al., 1982), suggesting similarity to that of methoxychlor. It is remarkable that such small differences in structures of DDT analogs affect the mechanism of activation. In vivo studies (Reuber, 1980) reveal that methoxychlor is a liver carcinogen in the mouse, rat, and possibly the dog and support the notion that the binding process described in this study represents a subcellular mechanism by which methoxychlor may express in vivo toxicity.4 However, it has been previously noted (Gillette, 1974a) that the binding of chemically reactive metabolites to macromolecules does not necessarily reflect toxicity. Clearly, it will be necessary to establish the in vivo binding of activated methoxychlor and to correlate this process with toxic activity. ACKNOWLEDGMENT This study was supportedby United StatesPublic Health Service Grant ES 00834 awarded by the National Institute of Environmental Health Sciences.

BULGER, TEMPLE,

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