Biotransformation and estrogenic activity of Methoxychlor and its metabolites in channel catfish (Ictalurus punctatus)

Marine Environmental 0 PII:

SO141-1136(97)00124-4

Research, Vol. 46, NO. 1-5, pp. 159-162, 1998 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-1136/98 $19.00+0.00

tLSEVlER

Biotransformation and Estrogenic Activity of Methoxychlor and its Metabolites in Channel Catfish (Zctalurus punctatus) D. Schlenk,“* D. M. Stresser,b J. Rimoldi,” L. Arcand,a J. McCantqn A. C. Nimrod” and W. H. BensoP “Department ofPharmacologyandEnvironmentalToxicology, EnvironmentalandCommunity Health Research Program, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi, USA bWorcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA cDepartment of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University, Mississippi, USA

ABSTRACT Methoxychlor (MXC) has been shown to possess estrogenic activity in mammals and fish. Although MXC does not appear to appreciably bind the mammalian estrogen receptor, its demethylated metabolites have been shown to be sign@antly more potent agonists and are believed responsible for estrogenic efsects in mammals following exposure to this pesticide. To determine whether cat$sh were capable of MXC demethylation, and, hence, activation to a more estrogenic compound, in vitro biotransformation studies were carried out using hepatic

microsomes from mature male channel catfish. Hepatic microsomes catalyzed the NADPH-dependent formation of monodemethylated (mono-MXC) and bisdemethylated (bis-MXC) metabolites of MXC. Treatment with mono-MXC at 40% qf the MXC dose in catfish significantly induced serum vitellogenin (Vg) levels compared to MXC. Estrogen receptor binding studies in catJsh liver cytosol showed that a racemic mixture of the mono-MXC had approximately 43 times the afinity for the receptor than MXC, but was still over lOOO-fold less potent that 17p-estradiol. These results demonstrate that catfish are capable of biochemically activating MXC to a more potent hepatic estrogen receptor agonist. 0 1998 Elsevier Science Ltd. All rights reserved

Methoxychlor is an organochlorine insecticide used to control a wide range of insect pests in field crops (Tomlin, 1994) and is considered to be a proestrogen in mammals (Bulger et al., 1978). The abnormal expression of the phospholipoprotein, vitellogenin (Vg) in mature male or juvenile animals, has been proposed as a biological indicator of estrogenic activity (Jobling et al., 1995). In order to bind the estrogen receptor in mammals, MXC *To whom correspondence

should be addressed. 159

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must first undergo demethylation to the mono- or bis-demethylated derivatives (Bulger et al., 1978). In rats, demethylation is catalyzed by several isoforms of cytochrome P450 monooxygenase including CYP 2Bl isoforms (Dehal and Kupfer, 1994). Although fish also possess multiple forms of P450, CYP 2B isoforms have not been identified (Stegeman and Hahn, 1994). Thus, the purpose of this study was to determine whether catfish are capable of converting MXC to metabolites that bind the estrogen receptor in catfish and/ or induce vitellogenesis. Microsomal fractions from livers dissected from sexually mature male channel catfish (Zctalurus punctatus) ranging from 250-300 g (14 months post-hatch) were isolated as previously described (Schlenk ef al., 1993). Incubations were conducted in a total volume of 250 ~1 containing 60 mM sodium phosphate buffer (pH 7.4), an NADPH regenerating system and 125pg microsomal protein with a range of substrate concentrations of [14C]MXC (1.8-3.2 Z&i pmol-I). After a 60min incubational period, the protein was precipiated with ethanol, and the supernatant evaporated under a stream of nitrogen. The residue was resuspended in 50 ~1 ethanol and applied to a TLC plate (C 18 reverse-phase) (Whatman, Inc., Clifton, NJ), which was developed in 75:24: 1 methanol:water:acetic acid. After drying, the chromatogram was subjected to quantitation with a System 200 Imaging Scanner (Bioscan, Inc., Washington, DC). The demethylated metabolites were identified by co-migration on TLC with authentic standards and metabolite derivization. Two radiolabeled metabolites of [14C]MXC were observed following incubation with NADPH (Fig. 1). Percent conversion was 8.92+ 1.37 in catfish with 87 and 13% of the metabolites coeluting with the mono- and bis-demethylated metabolites, respectively. The apparent Km for the microsomal demethylation of MXC to the mono-demethylated metabolite was 64.7 f 9.3 PM and the Vmax was 0.222 f 0.021 nmol mg-’ min’. Kinetic parameters for the bis-demethylated metabolite were not calculated. Monodemethylation of MXC leads to the formation of two enantiomeric metabolites in rats (Kurihara and Oku, 1991). Unfortunately, the enantiomers could not be chromatographically separated in this study. The affinities for the hepatic estrogen receptor of MXC and each metabolite were assessed by the ability of these compounds to displace 17/!?-[3H]-estradiol from specific binding sites in female catfish liver cytosol (Nimrod and Benson, 1997) (Fig. 2). The hydroxy-derivatives and MXC showed binding to the receptor with mono-MXC (I&e = 1.8 PM) having an approximately 43-fold higher affinity than the parent

Mono-OH-MXC

Bis-OH-MXC

;

lb

1;

LO

2;

migration (cm)

Fig. 1.

Metabolite profile of MXC following incubation with hepatic microsomes of male channel catfish in the presence of NADPH.

Methoxychlor and its metabolites in Ictalurus

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(I& = 78 PM). The ICsO values for bis-MXC and 17B-estradiol were 3.0 PM and 0.0017 PM, respectively. There were no steroselective differences between either the R or S-enantiomers of the mono-MXC (ICsOs= 3.0 PM). MXC exhibited weak to non-existent affinity for the estrogen receptor in the rat uterus, but mono-MXC showed significant binding, inhibiting estradiol binding with an ICsO of 0.6pM (Ousterhout et al., 1981). In the only reported study involving fish, MXC did not appear to bind the estrogen receptor in spotted seatrout (Thomas and Smith, 1993), but the demethylated metabolite was not examined. To assess the in vivo estrogenic activity of MXC and the racemic mixture of the monoMXC, catfish (n = 8) were separated into three groups for intraperitoneal injections. Group 1 received 1OOmgkg-’ mono-MXC; the second received 250mg kg-’ of MXC; and the third was injected 0.6 mg kg-i of 17p-estradiol. Injections were made on Days 1 and 3 with subsequent euthanasia on Day 6. Methods of Vg measurement (ELISA), MXC doses, and duration of exposures were determined from previous studies in channel catfish (Nimrod and Benson, 1996). Serum Vg was significantly increased in channel catfish treated with estradiol (55 000 & 24 000 pg ml-i) while only a slight increase was observed following MXC treatment (35 +0.57 pgml-I). No detectable Vg was observed in untreated animals. However, treatment of catfish with 100 mg kg-’ mono-(R,S)MXC led

Log competitor M

m=-501, -12

-11

-10

-9

-0

-7

-6

-5

-4

-3

Log competitor M Fig. 2. Inhibition of [3H]-estradiol binding to catfish liver cytosolic protein by 17/?-estradiol, MXC and MXC metabolites. Each value represents the mean of three replications.

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to nearly a 1OO-fold increase of Vg (1220 f 86 pg ml-‘) compared to vehicle treated control males. While a lOOO-fold difference was observed between estradiol and mono-MXC in receptor binding, only a 45fold difference was observed in Vg expression. The lack of correspondence between these measures suggests mono-MXC-mediated Vg induction may not be totally due to estrogen receptor binding and other mechanisms may be contributing to this estrogenic response. However, the enhanced affinity of the demethylated metabolite for the hepatic estrogen receptor in catfish and the significantly elevated levels of serum Vg following mono-MXC, but only minor elevation with MXC treatment, is consistent with MXC being a pro-estrogen, as observed in mammals and warrants further investigation.

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Stegeman, J. J. and Hahn, M. E. (1994) Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives, eds D. C. Malins and G. K. Ostrander, pp. 87-206. Lewis, Boca Raton, FL. Thomas, P. and Smith J. (1993) Marine Environmental Research 35, 1477151. Tomlin, C. (1994) The Pesticide Manual, 10th edn. Crop Protection Publications, Farnham, Surrey, UK.