Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene

Abstract

The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic monoand bis-demethylated metabolites (OH-MXC and HPTE respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2 mg MXC/kg daily for 6 days and sacrificed 24 hr after the last dose. The 3-MC treatment was a single 10 mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V max value (mean ± S.D., n=4) for glucuronidation of OH-MXC was 270 ± 50 pmol/ min/mg protein, higher than found for HPTE (110 ± 20 pmol/min/mg protein). For each substrate, the V max values observed in intestinal microsomes were approximately twice those found in the liver. The K m values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32 ± 0.04 mM for OH-MXC and 0.26 ± 0.06 mM for HPTE. The K m for the co-substrate, UDPGA, was higher in liver (0.28 ± 0.09 mM) than intestine (0.04 ± 0.02 mM). Treatment with 3-MC but not MXC increased the V max for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V max values for sulfonation of OH-MXC and HPTE respectively in liver cytosol were 7 ± 3 and 17 ± 4 pmol/min/mg protein and in intestinal cytosol were 13 ± 3 and 30 ± 5 pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.

The state of in vitro science for use in bioaccumulation assessments for fish

“…For example, subcellular fractions from the channel catfish liver and intestine were used to study the fate of methoxychlor, a pesticide shown to cause reproductive effects in fish. Methoxychlor was readily de‐methylated by microsomal cytochrome P450 to estrogenic metabolites [85], but the demethylated metabolites were much more slowly glucuronidated and sulfonated, suggesting the demethylated metabolites would bioaccumulate in the catfish [86]. Assuming other fish biotransform methoxychlor similarly to the channel catfish, these studies show why fish exposed to methoxychlor experience reproductive effects.…”

<i>In vitro</i> metabolism of pesticides and industrial chemicals in fish

“…About four times higher efficiency was observed in the intestinal microsomes of I. punctatus for the glucuronidation of mono-O-demethylated methoxychlor than in the hepatic ones, while cytosolic sulfonation of the di-O-demethylated metabolite proceeded at two times higher efficiency in the liver than in the intestine. 85) Significantly higher GST activity of this species against CDNB was observed in the liver than in the gills, and in the respective cytosol than in the microsomes. 86) As a result, more conjugates of chlorothalonil with GSH were formed in the hepatic cytosol.…”

“…54,55) These results show the usefulness of hepatocytes and liver slices to qualitatively estimate the in vivo metabolism of methoxychlor. In vitro studies using the hepatic microsomes (+ UDPGA) and cytosol (+ PAPS) of I. punctatus showed a formation rate of glucuronide conjugate ten-times higher than the sulfate one from the Odemethylated metabolite, 85) which partly explains the detection of glucuronides as dominant components in the bile and liver of the force-fed fish. 138) 3.1.3.…”

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass (Micropterus salmoides) liver

“…Please note that throughout the manuscript and in an effort to simplify, genes appear in italicized lower case letters and proteins appear in upper case letters. The liver was investigated because it is the major site for detoxification of compounds such as MXC and its metabolites (James et al, 2008) and is the site for the synthesis of VTG prior to translocation to the ovaries for incorporation into the eggs. Therefore, the liver has a major role in the normal reproductive process of female teleost fish of many egg-laying species and the liver can be responsive to estrogens in males after exposure by producing VTG.…”