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 mono- and 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 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/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+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/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+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). 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+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/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.

Figure 1

Pathways of MXC metabolism

Figure 2

TLC of ethyl acetate extracts and aqueous phases following incubation of hepatic microsomes with OH-MXC, no substrate or HPTE. The ethyl acetate extracts were evaporated to dryness under nitrogen and the residues reconstituted in ethanol as described in the methods section. Lane 1, ethyl acetate extract of an incubation with OH-MXC, showing the glucuronide conjugate; lane 2, β-glucuronidase-treated ethyl acetate extract from an incubation with OH-MXC, showing hydrolysis of the glucuronide conjugate; lane 3, aqueous phase from an incubation with OH-MXC, showing UDPGA but no glucuronide conjugate of OH-MXC; lane 4, ethyl acetate extract from an incubation with no substrate, showing a very faint band with similar retention to glucuronide conjugates; lane 5, aqueous phase from incubations with no substrate, showing only unreacted UDPGA; lane 6, ethyl acetate extract from an incubation with HPTE, showing the glucuronide conjugate; lane 7, β-glucuronidase-treated ethyl acetate extract from an incubation with HPTE, showing hydrolysis of the glucuronide; lane 8, aqueous phase from an incubation with HPTE, showing unreacted UDPGA but no glucuronide conjugate.

Figure 3

Glucuronidation of endogenous components in microsomes. Microsomes were incubated with ¹⁴C-UDPGA in the absence of exogenous aglycone. Compared with microsomes from control catfish, microsomes from MXC-treated catfish showed higher formation of glucuronides in the absence of exogenous substrate, p<0.05. Microsomes from 3-MC-treated fish were not different from controls, p>0.05.

Figure 4

HPLC of extracts of microsomes, with UV₂₄₅ detection. Panel A shows an extract from control microsomes, panel B an extract of microsomes from MXC-exposed fish and panel C an extract of control microsomes spiked with standards for MXC, OH-MXC and HPTE.

Figure 5

Radiochromatogram showing TLC separation of the products of incubation of ³⁵S-PAPS with hepatic cytosol in the presence and absence of exogenous substrate. Arrows indicate the sulfate conjugates. Lane 1 shows sulfate conjugates formed from endogenous compounds present in hepatic cytosol; lane 2 shows the sulfate conjugate formed by incubation of hepatic cytosol with OH-MXC, 800 μM; lane 3 shows sulfate conjugates formed in the presence of OH-MXC, 50 μM, and hepatic cytosol; and lane 4 shows sulfate conjugates formed by incubation HPTE, 100 μM, with hepatic cytosol and ³⁵S-PAPS.

Figure 6

Inhibition of glucuronidation of 3-OH-BaP by OH-MXC in hepatic microsomes from control catfish. The sigmoidal dose-response curve shown is typical of results with catfish hepatic microsomes.