Influence of β-Naphthoflavone and Methoxychlor Pretreatment on the Biotransformation and Estrogenic Activity of Methoxychlor in Channel Catfish (Ictalurus punctatus)

doi:10.1006/taap.1997.8194

Toxicology and Applied Pharmacology

Volume 145, Issue 2, August 1997, Pages 349-356

https://doi.org/10.1006/taap.1997.8194 Get rights and content

Abstract

The organochlorine pesticide methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane] (MXC) has been classified as a proestrogen in mammals and fish, requiring demethylation prior to eliciting estrogenic activity or binding to the estrogen receptor. While microsomal demethylation occurs readily in the liver of fish, little is known about the enzyme(s) responsible or the effect of cytochrome P450 (CYP) inducers, other than those of CYP1A and CYP2K, on biotransformation. Consequently, male channel catfish were pretreated with MXC or β-naphthoflavone (BNF), alone and in combination, to determine their effects on CYP protein expression, MXC biotransformation by hepatic microsomes, microsomal protein binding, and MXC estrogenic activity as determined by serum vitellogenin and 17β-estradiol. Liver microsomes of both treated and untreated mature male catfish catalyzed formation of monodemethylated MXC, bisdemethylated MXC, as well as ring-hydroxylated metabolites. Pretreatment with BNF did not affect MXC metabolite profiles, overall rates of MXC biotransformation, or microsomal proteins recognized by anti-trout CYP2K1, but had the expected effect of inducing CYP1A and associated ethoxyresorufinO-deethylase activity. By contrast, pretreatment with MXC, alone or in combination with BNF, significantly reduced rates of MXC biotransformation and binding to liver microsomal protein. MXC/BNF cotreatment followed by MXC significantly induced serum vitellogenin, whereas MXC treatment alone led to a nonsignificant increase in vitellogenin and a significant increase in serum 17β-estradiol. Thus, estrogenic activity elicited by cotreatment with MXC and BNF can occur despite diminished capacity of liver microsomes to catalyze formation of estrogenic demethylated metabolites or metabolites that bind microsomal protein. Possible mechanisms of MXC-induced attenuation of CYP-dependent metabolism are discussed.

References (32)

D.E. Buhler et al.
The role of biotransformation in the toxicity of chemicals
Aquat. Toxicol.
(1988)
W.H. Bulger et al.
Studies on thein vivoin vitro
Biochem. Pharmacol.
(1978)
L.E. Gray et al.
A dose–response analysis of methoxychlor-induced alterations of reproductive development and function in the rat
Fundam. Appl. Toxicol.
(1989)
I.A. Khan et al.
Disruption of neuroendocrine function in Atlantic croaker exposed to arochlor 1254
Mar. Environ. Res.
(1996)
D. Kishimoto et al.
Enantiotopic selectivity of cytochrome P450-catalyzed oxidative demethylation of methoxychlor: Alteration of selectivity depending on isozymes and substrate concentrations
Pestic. Biochem. Physiol.
(1995)
P.M. Loveland et al.
Identification and mutagenicity of aflatoxicol M1 produced by metabolims of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout fed beta-naphthoflavone
Food Chem. Toxicol.
(1983)
O.H. Lowry et al.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
(1951)
A.C. Nimrod et al.
Estrogenic responses to xenobiotics in channel catfish (Ictalurus punctatus
Mar. Environ. Res.
(1996)
D. Schlenk et al.
Channel catfish liver monooxygenases: Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases
Biochem. Pharmacol.
(1993)
D.M. Stresser et al.
Ring hydroxylation of [o³
Anal. Biochem.
(1996)

D.E. Williams et al.

Sex differences in cytochrome P450 isozyme composition and activity in kidney microsomes of mature rainbow trout

Biochem. Pharmacol.

(1986)

J.C. Wingfield et al.

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding

Steroids

(1975)

L.-X. Zhou et al.

Cytochrome P450 catalyzed covalent binding of methoxychlor to rat hepatic, microsomal iodothyronine 5′-monodeiodinase, type I: Does exposure to methoxychlor disrupt thyroid hormone metabolism?

Arch.Biochem. Biophys.

(1995)

C.K. Atterwill et al.

Endocrine Toxicology

(1992)

R. Binder et al.

Induction of cytochrome P-450 dependent monooxygenase systems in embryos and eleutheroembryos of the killifishFundulus heteroclitus

Chem.–Biol. Interact.

(1985)

Cited by (41)

Effects of the pesticide methoxychlor on gene expression in the liver and testes of the male largemouth bass (Micropterus salmoides)
2008, Aquatic Toxicology
The organochlorine pesticide methoxychlor (MXC) is an environmental estrogen known to stimulate the expression of the egg-yolk protein, vitellogenin (Vtg) in fish species. To begin to understand the underlying mechanisms for how MXC exerts its deleterious effects on the endocrine system, male largemouth bass (Micropterus salmoides) were treated with 2.5, 10, or 25 mg/kg MXC and compared to fish pair-treated with 1 mg/kg 17β-estradiol (E₂), and vehicle control. Fish were sacrificed 24, 48, or 72 h following treatment. The liver and testes were then assayed for changes in expression of the three bass estrogen receptors (ERsα, βa, and βb) in tissues, as well as Vtg and cytochrome P450 (CYP) 3A isoform 68 in the liver and steroidogenic acute regulatory protein (StAR) in the testes. In the liver, significant increases in gene expression were seen for each of the genes measured by 24 h and each returned to the level of the vehicle by 72 h. Total testosterone 6β-hydroxylase activity, reflective of CYP3A activity, was also increased by 24 h for all of the exposures. In the testes, ERα was unaffected by any treatment, ERβa was up-regulated only by MXC, peaking at 24 h for the 2.5 and 10 mg/kg MXC and at 48 h for the 25 mg/kg MXC treatment. By 72 h, the MXC effects had disappeared, while E₂ significantly decreased the expression of ERβa mRNA. ERβb expression in the testes was stimulated by all concentrations of MXC by 24 h and the effect remained up to 72 h, whereas E₂ had no effect. Finally, StAR expression was also found to be decreased by E₂ and all MXC treatments. However, the effect on StAR expression by E₂ occurred within 24 h, while the effect by all concentrations of MXC was not seen until 72 h after treatment. The stimulatory effects of E₂ and 25 mg/kg MXC on the expression of the ERs in the liver were opposite to the responses seen in the testes, suggesting an inverted relationship between these two tissue types. These results provide a possible mechanism showing that alterations in reproductive signaling in male fish by xenoestrogens not only increase Vtg expression in the liver, but may also decrease reproductive success by muting some of the estrogen signals required for sperm production.
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
2008, Aquatic Toxicology
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 2 mg MXC/kg daily for 6 days and sacrificed 24 h 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.
Are steroids really the cause for fish feminization? A mini-review of in vitro and in vivo guided TIEs
2008, Marine Pollution Bulletin
Feminization of fish has been reported throughout the world in freshwater and marine systems. While the population impacts are conflictive, enough negative effects warrant additional research into causation. In order to ascertain the identities of specific feminizing agents, variants of toxicity identification evaluations (TIEs) have been employed. The majority of these evaluations have utilized in vitro estrogen receptor-based cell-lines to identify chromatographic fractions that possess biological activity from predominately wastewater derived from municipal treatment facilities and have concluded that synthetic and natural estrogens are the primary cause for feminization of fish. This paper will focus on three aquatic systems impacted by wastewater originating from purely domestic, and industrial/domestic secondary treatment systems. Wastewater and sediment extracts were evaluated by in vitro and in vivo biological responses in a TIE fractionation design. While in vitro responses tended to mirror in vivo responses in purely domestic wastewater systems, in vitro responses tended to severely underestimate in vivo estrogenic activity when normalized to estradiol equivalents in more complex systems. TIE fractionation schemes using in vivo biological responses failed to indicate any relationship to steroids in either wastewater or sediment extractions. These data consistently support the view that mechanisms other than direct ER binding and activation by toxicants may be important in the feminization of fish particularly residing in habitats that receive complex wastewater or agricultural effluents.
A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions
2007, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
Scientific literature from the past 25 years was searched to obtain in vitro biotransformation rate and affinity data for fish. To maximize the environmental relevance of this dataset, we focused on studies conducted at multiple substrate concentrations, and established acceptance criteria with respect to assay temperature and pH. Altogether, enzyme rate and affinity parameters are provided for 43 species and 77 compounds. In all but three instances, the reported reactions exhibited saturation at high substrate concentrations and could be used to calculate Michaelis–Menten rate (V_max) and affinity (K_m) constants. Most of this information was obtained using in vitro systems derived from liver tissue. Information from non-hepatic tissues was included, however, to provide a basis for comparisons among tissues. Where possible, in vitro enzyme parameters were examined to compare: (1) hepatic metabolism of a common substrate within a species, (2) hepatic metabolism of common substrates by different species, and (3) metabolism of a common substrate by different tissues of one species. Comparisons within species highlight a number of factors that may substantially influence xenobiotic metabolism in fish including gender, life stage, and acclimation temperature. Limited data suggest that V_max and K_m for the same reaction may vary by up to three orders of magnitude among species.
Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances
2006, Aquatic Toxicology
Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.
Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)
2006, Chemico-Biological Interactions
Citation Excerpt :
However, little is known about the effects of xenoestrogens on CYP1A. For example, CYP1A catalytic activity was suppressed when medaka exposed to EE2 [21], while EROD activity were evaluated in adult male channel catfish exposed to methoxychlor [30]. In addition, these similar results were observed in effluent exposure fish [21].
Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 μg/l and decreased in male and female animals exposed to 200 μg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 μg/l, but decreased in the 200 μg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 μg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 μg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 μg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 μg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.

View all citing articles on Scopus

^☆: D. C. MalinsG. K. Ostrander, Eds.

¹: To whom correspondence should be addressed.

View full text

Article preview

Toxicology and Applied Pharmacology

Abstract

References (32)

The role of biotransformation in the toxicity of chemicals

Aquat. Toxicol.

Studies on thein vivoin vitro

Biochem. Pharmacol.

A dose–response analysis of methoxychlor-induced alterations of reproductive development and function in the rat

Fundam. Appl. Toxicol.

Disruption of neuroendocrine function in Atlantic croaker exposed to arochlor 1254

Mar. Environ. Res.

Enantiotopic selectivity of cytochrome P450-catalyzed oxidative demethylation of methoxychlor: Alteration of selectivity depending on isozymes and substrate concentrations

Pestic. Biochem. Physiol.

Identification and mutagenicity of aflatoxicol M1 produced by metabolims of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout fed beta-naphthoflavone

Food Chem. Toxicol.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

Estrogenic responses to xenobiotics in channel catfish (Ictalurus punctatus

Mar. Environ. Res.

Channel catfish liver monooxygenases: Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases

Biochem. Pharmacol.

Ring hydroxylation of [o³

Anal. Biochem.

Sex differences in cytochrome P450 isozyme composition and activity in kidney microsomes of mature rainbow trout

Biochem. Pharmacol.

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding

Steroids

Cytochrome P450 catalyzed covalent binding of methoxychlor to rat hepatic, microsomal iodothyronine 5′-monodeiodinase, type I: Does exposure to methoxychlor disrupt thyroid hormone metabolism?

Arch.Biochem. Biophys.

Endocrine Toxicology

Induction of cytochrome P-450 dependent monooxygenase systems in embryos and eleutheroembryos of the killifishFundulus heteroclitus

Chem.–Biol. Interact.

Cited by (41)

Effects of the pesticide methoxychlor on gene expression in the liver and testes of the male largemouth bass (Micropterus salmoides)

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

Are steroids really the cause for fish feminization? A mini-review of in vitro and in vivo guided TIEs

A compilation of in vitro rate and affinity values for xenobiotic biotransformation in fish, measured under physiological conditions

Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Regular ArticleInfluence of β-Naphthoflavone and Methoxychlor Pretreatment on the Biotransformation and Estrogenic Activity of Methoxychlor in Channel Catfish (Ictalurus punctatus)☆

Abstract

Aquat. Toxicol.

Biochem. Pharmacol.

Fundam. Appl. Toxicol.

Mar. Environ. Res.

Pestic. Biochem. Physiol.

Food Chem. Toxicol.

J. Biol. Chem.

Mar. Environ. Res.

Biochem. Pharmacol.

Anal. Biochem.

Biochem. Pharmacol.

Steroids

Arch.Biochem. Biophys.

Endocrine Toxicology

Induction of cytochrome P-450 dependent monooxygenase systems in embryos and eleutheroembryos of the killifishFundulus heteroclitus

Chem.–Biol. Interact.

Regular Article
Influence of β-Naphthoflavone and Methoxychlor Pretreatment on the Biotransformation and Estrogenic Activity of Methoxychlor in Channel Catfish (Ictalurus punctatus)☆