Environmental Fate and Safety Management of Agrochemicals

Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: March 8, 2005 | doi: 10.1021/bk-2005-0899.ch016

Chapter 16

Comparative In Vitro Metabolism of [ C]Methoxychlor in Vertebrate Species Using Precision-Cut Liver Slice Technique 14

K. Ohyama Metabolism Laboratory-I, Chemistry Division, Institute of Environmental Toxicology, Ibaraki 303-0043, Japan

Introduction The organochlorine insecticide methoxychlor (MXC) [1,1,1-trichloro-2,2bis(4-methoxyphenyl) ethane] is a structural analogue of the environmentally persistent compound DDT. Although MXC is biodegradable (1-3) and it exhibits relatively lower acute toxicity in mammals compared with DDT (1), various studies have shown that methoxychlor may elicit estrogenic responses in mammals (4,5) and also wildlife including fish (6,7). Such estrogenic effects of methoxychlor are believed to be caused by the action of its demethylated metabolites (8-10), which are major phase I metabolites catalyzed by the cytochrome P450 monooxygenase system. Therefore, it is important to understand the metabolism of methoxychlor in mammals and also wildlife species. Most of the studies, however, have focused on the phase I metabolic reaction (functionalization reaction) of methoxychlor, but little information is available for methoxychlor phase II metabolism (conjugation reaction). Since the precision-cut tissue slice technique using an automated mechanical

© 2005 American Chemical Society In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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186


tissue slicer (e.g. Krumdieck Tissue Slicer) was introduced, it has been applied to drug and xenobiotic metabolism and toxicity studies in recent years (11-13). The slice of liver tissue contains many intact cell layers which maintain hepatic architecture and cell-to-cell interactions, therefore liver slices are expected to provide integrated profiles of phase I and phase II metabolites that would be more similar to in vivo metabolism than other in vitro models. This paper describes comparative in vitro metabolism of methoxychlor by precision— cut liver slices from rat, mouse, Japanese quail and rainbow trout.

In vitro metabolism of methoxychlor by precision-cut rat, mouse, Japanese quail and rainbow trout liver slices The liver slices of male rats (Sprague-Dawley, 9 weeks old), male mice (CD-1, 18-19 weeks old), male Japanese quail (WE strain, 100-130 g body wt.) and juvenile rainbow trout (Oncorhynchus mykiss, 100-250g body wt.) were prepared using the Krumdieck Tissue Slicer (Alabama Research & Development) in ice chilled Krebs-Henselite buffer (containing 20mM fructose, pH 7.4) (14). The slices were cultured in Krebs-Henselite buffer containing 5 μΜ of [ring-U- C]methoxychlor (5.84 MBq/mg) in 12-well plates (15) with continuous gyratory shaking under air/C0 (95/5) atmosphere (1 slice/1 mL medium/well). Metabolites in the cultured medium and slice extracts were analyzed by a reversed phase radio-HPLC and LC/ESI/MS/MS. Validity of the test preparation was evaluated by monitoring a reaction of 7-ethoxycumarine metabolism (12,16), forming 7-hydroxycoumarin and its sulfate and glucuronide conjugates, concurrently with each experimental run. In precision-cut liver slice preparation from all test animal species, timedependent metabolism of methoxychlor was observed, and quantitative and qualitative differences were detected (17). Representative HPLC profiles of methoxychlor metabolites after metabolic reaction are shown in Figures 1 and 2. In rat preparations (Figure 1), a bis-demethylated compound (bis-OH-MXC) and its O-glucuronide were the major metabolites. The glucuronide conjugate of mono-OH-MXC (mono-demethylated methoxychlor) was not found, even though trace amounts of the mono-OH-MXC was detected as the transient intermediate at shorter incubation times. Therefore, methoxychlor was appeared to be quickly metabolized to bis-OH-MXC by sequential O-demethylation, followed by subsequent O-glucuronidation. A very polar metabolite, bis-OHM X C 4-0-sulfate 4'-0-glucuronide, was also formed as the rat-specific metabolite. On the other hand, mono-OH-MXC glucuronide was the major metabolite for mouse and Japanese quail, and bis-OH-MXC glucuronide was observed only as the minor one, so that mono-0-demethylation and subsequent O-glucuronidation may be considered as the main metabolic reactions for both 14

2

In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

187

b i s - O H - M X C glucuronide b l s - O H - M X C 4-O-SUlfate ^-Oalucuronlde

bis-OH-MXC

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1 Τ

methoxychlor mono-OH-MXC mono-un M X C

ί

1

extracts

Medium


L..K

10

Min

15

20

, ...

u

25

Figure 1. Representative radio-HPLC chromatograms of in vitro metabolism of [ C]methoxychlor by precision-cut rat liver slices. The chromatograms show the radioactive components detected in the slice extracts after 1-hr incubation and in cultured medium after 4-hr incubation. 14

dechlorinated m o n o - O H - M X C glucuronide ι m o n o - O H - M X C glucuronide

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1 b i s - O H - M X C glucuronide

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.

i

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..

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methoxychlor 1

T

*

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.À

A.

1

0>5Q 0

ο

Ι

Mouse

. Trout

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3(

τ

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5

10

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15

20

25

Figure 2. Representative radio-HPLC chromatograms of in vitro metabolism of [ C]methoxychlor by precision-cut mouse, Japanese quail and rainbow trout liver slices. The chromatograms show the radioactive components detected in the cultured medium after metabolic reaction. 14



188 species. The reductively dehalogenated metabolite, dechlorinated mono-OHMXC, was uniquely observed only in mouse preparation. In the case of trout, nearly equal amounts of mono- and bis-OH-MXC glucuronide were produced and unconjugated forms of those metabolites were detected only as minor products. It seems dual metabolic reactions, involving demethylation and glucuronidation, were equally contributed for metabolism of the intermediate metabolite, mono-OH-MXC, for this particular species. Figure 3 shows the ratio of (mono-OH-MXC)/(mono- + bis-OH-MXC), where the values of mono- and bis- demethylated metabolites also include the amounts of their corresponding conjugates. After reaction in each animal preparation, more than 80% of demethylated metabolites were detected as a mono-demethylated form for mouse and quail, about 50% for trout, and less than 5 % for rat. The results indicate that O-demethylase enzyme(s) in rat can easily remove both methyl groups from the parent molecule, but only one methyl group can be preferably demethylated by the enzyme(s) in mouse and quail. Rainbow trout produces almost equal amounts of mono- and bis- demethylated metabolites. These facts suggest that there are species differences in the manner of phase I oxidative demethylation, and such differences are probably due to the

1.00

Incubation time (hr)

Figure 3. Ratio of mono- and bis-demethylated metabolites of methoxychlor formed by precision-cut rat, mouse, Japanese quail and rainbow trout liver slices. The amounts of corresponding glucuronide conjugate are also included in this analysis.


189 differences in the reactivity of the contributing P450s enzymes toward methoxychlor and its mono-demethylated metabolite.


Stereoselective formation of the mono-hydroxy metabolite of methoxychlor Methoxychlor is a prochiral compound, and the mono-demethylation reaction would yield chiral metabolites, (/?)- and (S)-mono-OH-MXC (Figure 4). Understanding the nature of the P450 enzymes contributing for the demethylation reaction of methoxychlor in different animal species, stereoselectivity of the initial demethylation step, which is the common metabolic reaction for all animal species tested, was further investigated. Figure 5 shows the ratio of (/?)- and (S)-mono-OH-MXC formed in methoxychlor metabolism. The amounts of corresponding conjugates are also included in this analysis. The enantiomers of the mono-demethylated metabolite were separated by chiral HPLC and identified by HPLC retention time comparison with authentic standards. Rat and mouse selectively formed (5)mono-OH-MXC enantiomer at approximately 90 and 75 % yield, respectively. In contrast, quail and trout produced (/?)-mono-OH-MXC at more than 85 % yield. The results indicate that the oxidative mono-demethylation reaction occurs stereo-selectively and different animal species exhibit different enantioselectivity, suggesting that contributing P450 can recognize the stereological conformation of methoxychlor and that there are species differences in such molecular recognizability.

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HO,

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3

3

Oxidative demethylation

(S)-Mono-OH-MXC


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190


•

Rat

Mouse

(S)-mono-OH-MXC

Quail

Trout

Figure 5. Enantiotopic selectivity in the oxidative mono-demethylation of methoxychlor by precision-cut rat, mouse, Japanese quail and rainbow trout liver slices. The amounts of corresponding conjugates are also included in this analysis.

Summary and conclusions In the present study, precision-cut liver slices were used to investigate the metabolism of methoxychlor. The incubation of [ C]methoxychlor with rat, mouse, Japanese quail and rainbow trout liver slices resulted in time-dependent metabolism, forming mono- and/or bis- demethylated metabolites. In addition, their corresponding phase II metabolites were subsequently formed as the final products. These metabolites were essentially those already reported as the major methoxychlor in vivo metabolites in mammals (1-3), therefore the precision-cut liver slice technique is shown to be a useful model to study biotransformations of xenobiotics. By comparing the metabolic profiles obtained from different animal species in detail, clear differences are observed, especially in the pattern of phase I metabolism. Rats showed intensive sequential 0-demethylation forming bis-demethylated metabolites, in contrast, mouse and quail produce mostly mono-demethylated products. Rainbow trout can equally produce both monoand bis-demethylated metabolites. It appears that the nature of the cytochrome P450 enzymes involved in the oxidative demethylation reaction are vary in different animal species. The enzymes in quail and mouse preferably remove only one methyl group and the enzyme in rat can easily remove both methyl groups. In addition, such a demethylation reaction proceeds enantioselectively 14



191 and is also species dependent. The rodent species, rat and mouse, form (S)mono-OH-MXC, preferentially, and quail and trout selectively produce the (R)isomer. Species differences in the metabolism of xenobiotics can be basically explained by the qualitative and quantitative differences of expressed enzymes contributing to the metabolic reactions. Among such enzymes, the cytochrome P450 monooxygenase system plays important roles in the oxidation of structurally diverse compounds, such as drugs and xenobiotics, as well as endogenous compounds. P450 enzymes consist of multiple isoforms, and such multiplicity causes structural and functional diversity of this enzyme. The mechanistic phase I metabolism of methoxychlor by P450s is already well studied and it is known that oxidative O-demethylation is the predominant reaction (18-20). Hu and Kupfer (21) indicated CYP1A2, 2C9, and 2C19 isoforms were likely the major enzymes contributing to methoxychlor metabolism by using cDNA expressed human P450s. Among those CYPs, CYP2C19 and 1A2 contributed to the catalytic reactions forming both monoand bis-demethylated metabolites and 2C9 mainly contributed to an initial monodemethylation path. Enantioselectivity of the mono-demethylation reaction by human liver microsomes and P450 isoforms has also been demonstrated (22). The (5)-mono-OH-MXC was the major enantiomer produced by CYP2C9, 2C19 and human liver microsomes, and CYP1A2 preferably forms (/?)-mono-OHMXC. Kishimoto et al. (23) demonstrated that rat CYP2C6 and 2A1 isolated from non-induced male rats, exhibited enantiotopic selectivity of methoxychlor O-demethylation forming (5)-mono-OH-MXC, selectively. They additionally showed, in contrast that rat 2B1 and 2B2, isolated from Phenobarbital-induced male rats, which also exhibited methoxychlor 0-demethylase activity, had lower enantiotopic selectivity. These results indicate that there is a different substrate specificity in the different CYPs species contributing for methoxychlor Odemethylation and stereostructural preference may be involved in such substrate specificity. Differences in the substrate specificity of contributing P450s in different animal species may provide one possible explanation for the differences in methoxychlor metabolic profiles observed in the present study. The metabolic pathways including stereochemistry of methoxychlor metabolism by liver slices obtained in this study are proposed in Figure 6. When the metabolic pathways are expressed in 2-dimentional molecular structures, mouse and quail show quite similar patterns, since the oxidative mono-Odemethylation and subsequent glucuronidation are the main metabolic pathways for both species. However, once the stereological structures are taken into account, the metabolic pathways in these two species turn out to be different, because of the enantioselectivity of the mono-demethylation reactions. Indeed, when the stereological structures are taken into consideration, it is shown that the metabolic pathways of methoxychlor from the four test animal species are all



192 different. Superficially, the reactions involved in methoxychlor metabolism appear relatively simple, since oxidative demethylation and subsequent conjugation are the main processes in most cases. Even in such simple transformations, it has been shown that diverse metabolic reactions in different animal spices are involved. From the toxicological point of view, the different metabolic profiles of methoxychlor observed in different animal species may need to be considered to understand the metabolism-induced methoxychlor toxicity in individual animal species. In the case of mouse and quail, only trace amounts of bis-OH-MXC, which is believed to be the most active metabolite causing methoxychlor induced estrogenic responses (4, 8-10% were detected, therefore, the toxic activity of the main metabolite, mono-OH-MXC and its 0-glucuronide, also need to be considered. As for enantiotopic metabolites, it would be also important to understand if there is a difference in toxicological activity between (R)- and (5)isomers. If marked toxicological difference is observed between these two isomers, the toxicity induced by stereoselective metabolism may need to be further considered.

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Conjugation

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H* CHCI2

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DeCI-monp-OH-MXC

Bis-OH-MXC [Trout] ^ [Rat]

.

[Mouse] Conjugation

[Mouse]

1

Conjugation

Figure 6. Proposed metabolic pathways of methoxychlor by precision-cut rat, mouse, Japanese quail and rainbow trout liver slices.


193 Additionally, all phase I metabolites were extensively conjugated with Dglucuronic acid and most metabolites detected after longer incubation, were conjugated forms in any test animal species, indicating that phase II reactions also play important roles for metabolism of methoxychlor. Therefore, contribution of phase II metabolism which may (or possibly may not) work as a detoxification process, needs to be taken into account to explain the in vivo toxicity of methoxychlor.


Acknowledgements The author is grateful to Dr. Norio Kurihara kindly providing authentic (R)and (S)-mono-hydroxy methoxychlor standards and for his helpful advice. The author acknowledges Dr. K. Kato, Dr. K. Sato and Mr. S. Maki for their valuable suggestions. I also would like to thank current and past colleagues at the Institute of Environmental Toxicology for their discussions and technical assistance. Part of this study was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan.

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7. 8. 9.

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194 10. Elsby R.; Maggs J. L.; Ashby J.; Paton D.; Sumpter J. P.; Park B. K. J. Pharmacol. Exp. Ther. 2001, 296, 329-337. 11. Ekins S. Drug Metab. Rev. 1996, 28, 591-623. 12. Steensma Α.; Beamand J. Α.; Walters D. G.; Price R. J.; Lake B. G. Xenobiotica 1994, 24, 893-907. 13. Singh Y.; Cooke J. B.; Hinton D. E.; Miller M . G. Drug Metab. Dispos. 1996, 24, 7-14. 14. Dogterom P. Drug Metab. Dispos. 1993, 21, 699-704. 15. Hashemi E.; Dobrota M.; Till C.; Ioannides C. Xenobiotica 1999, 29, 11-25. 16. Walsh J. S.; Patanella J. E.; Halm Κ. Α.; Facchine K. L. Drug Metab. Dispos. 1995, 23, 869-874. 17. Ohyama K.; Maki S.; Sato K.; Kato. Xenobiotica 2004, unpublished. 18. Dehal S. S.; Kupfer D.; Drug Metab. Dispos. 1994, 22, 937-946. 19. Stresser D. M.; Kupfer D. Drug Metab. Dispos. 1998, 26, 868-874. 20. Kurihara N.; Oku A. Pestic. Biochem. Physiol. 1991, 40, 227-235. 21. Hu Y.; Kupfer D. Drug Metab. Dispos. 2002, 30, 1035-1042. 22. Hu Y.; Kupfer D. Drug Metab. Dispos. 2002, 30, 1329-1336. 23. Kishimoto D.; Oku Α.; Kurihara N.; Pestic. Biochem. Physiol. 1995, 51, 1219.


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