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Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p′-DDD in Y-1 adrenal cells
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
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2007 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 81, no 11, 793-801 p.Article in journal (Refereed) Published
Abstract [en]

The environmental pollutant 3-MeSO2–DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p′-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO2–DDE and o,p′-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO2–DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p′-DDD reached similar levels of binding as 3-MeSO2–DDE. The binding of o,p′-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO2–DDE, but not of o,p′-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO2–DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO2–DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO2–DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO2–DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO2–DDE and o,p′-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice in vivo. This reveals a notable in vitro/in vivo discrepancy, the contributing factors of which remain unexplained. We consider the Y-1 cell line as appropriate for studies of the cellular mechanisms behind the adrenocortical toxicity of these substances.

Place, publisher, year, edition, pages
2007. Vol. 81, no 11, 793-801 p.
Keyword [en]
Adrenal cortex, Cytochrome P450, Metabolic activation, Toxicity, Y-1
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-14330DOI: 10.1007/s00204-007-0206-5ISI: 000250537400006PubMedID: 17487473OAI: oai:DiVA.org:uu-14330DiVA: diva2:42100
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2017-12-11Bibliographically approved
In thesis
1. In Vitro Studies of Adrenocorticolytic DDT Metabolites, with Special Focus on 3-methylsulfonyl-DDE
Open this publication in new window or tab >>In Vitro Studies of Adrenocorticolytic DDT Metabolites, with Special Focus on 3-methylsulfonyl-DDE
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The DDT metabolite 3-methylsulfonyl-DDE (3-MeSO2-DDE) is bioactivated by cytochrome P450 11B1 (CYP11B1) in the adrenal cortex of mice and forms irreversibly bound protein adducts, reduces glucocorticoid secretion, and induces cell death selectively in cortisol-producing adrenocortical cells. 3-MeSO2-DDE has therefore been proposed as a lead compound for an improved adrenocortical carcinoma (ACC) therapy.

The aims of this thesis were to (1) develop in vitro test systems based on murine and human adrenocortical cell lines and to (2) investigate the mechanisms behind 3-MeSO2-DDE toxicity in adrenocortical cells.

The cytotoxic and endocrine-modulating effects of 3-MeSO2-DDE were compared to those of o,p′-DDD (mitotane), the current ACC therapy, and to those of several structurally analogous compounds in both murine and human cell lines. 3-MeSO2-DDE bioactivation and cytotoxicity proceeded in a similar manner in the murine adrenocortical Y-1 cell line as in mice in vivo. The effects were highly structure-specific. Moreover, 3-MeSO2-DDE formed irreversibly bound protein adducts and caused cell death also in the human H295R cell line, and was slightly more cytotoxic than o,p′-DDD. However, 3-MeSO2-DDE toxicity in human cells was not affected by the CYP11B1 inhibitor etomidate, suggesting that bioactivation in human cells is performed by additional/other enzyme(s) than CYP11B1. 3-MeSO2-DDE generated biphasic responses in cortisol and aldosterone secretion and in expression levels of the steroidogenic genes CYP11B1, CYP11B2, and StAR. Such hormesis-like responses were not seen for o,p′-DDD or the precursor DDT metabolite p,p′-DDE.

In addition, the two o,p′-DDD enantiomers (R)-(+)-o,p′-DDD and (S)-(-)-o,p′-DDD exhibited slight differences in cytotoxic and endocrine-modulating activity in H295R cells.

In conclusion, this thesis  provides  extended  knowledge  on  the  mechanisms  of  action  of 3-MeSO2-DDE and points out important differences in effects between murine and human cells. Lead optimisation studies of 3-MeSO2-DDE using the herein presented in vitro test systems are ongoing.

 

Place, publisher, year, edition, pages
Uppsala, Sweden: Acta Universitatis Upsaliensis, 2010. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 744
Keyword
Toxicity, 3-methylsulfonyl-DDE, CYP, adrenal cortex, metabolic activation, steroid hormones, Y-1, H295R, mitotane, adrenocortical carcinoma
National Category
Pharmacology and Toxicology
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-122721 (URN)978-91-554-7808-7 (ISBN)
Public defence
2010-06-01, Zootissalen, Evolutionsbiologiskt centrum (EBC), Villavägen 9, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2010-05-11 Created: 2010-04-16 Last updated: 2018-01-12Bibliographically approved
2. Adrenocorticolysis Induced by 3-MeSO2-DDE: Mechanisms of Action, Kinetics and Species Differences
Open this publication in new window or tab >>Adrenocorticolysis Induced by 3-MeSO2-DDE: Mechanisms of Action, Kinetics and Species Differences
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The DDT metabolite 3-methylsulphonyl-DDE (3-MeSO2-DDE) induces cell death specifically in the adrenal cortex of mice after a cytochrome P45011B1 (CYP11B1)-catalysed bioactivation. This substance is not only an environmental pollutant, but also a suggested lead compound for an improved chemotherapy of adrenocortical carcinoma (ACC). The aim of the thesis was to further investigate this compound in terms of kinetics, cell death mechanisms and species differences. The pharmacokinetics of 3-MeSO2-DDE and the current drug for ACC, o,p’-DDD, was studied during 6 months following a single dose in minipigs. The elimination was slower for 3-MeSO2-DDE than for o,p’-DDD, indicated by a lower clearance and longer t½ in plasma and subcutaneous fat. Both substances remained in fat tissue during the whole study period. Unlike o,p’-DDD, 3-MeSO2-DDE was retained also in liver. The adequacy of the murine adrenocortical cell line Y-1 was evaluated for studies of adrenotoxic compounds. The Y-1 cells proved to be an appropriate test system for future mechanism studies, since CYP-catalysed irreversible binding, inhibited corticosterone production induced by 3-MeSO2-DDE and o,p’-DDD were successfully demonstrated. Cell death of 3-MeSO2-DDE in the mouse adrenal cortex was implied to be necrotic. Early apoptotic signalling (i.e. up-regulation of caspase-9) was observed, although it seemed to be interrupted by ATP-depletion and anti-apoptotic actions by heat shock protein 70, resulting in lack of activation of caspase-3. Using cultured adrenal tissue slices, two not previously studied species were examined ex vivo regarding adrenal binding of 3-MeSO2-[14C]DDE. Binding was found in the hamster adrenal cortex and in assumed cortical cells in the medulla, while the guinea pig adrenal was devoid of binding. This emphasises the species specificity in bioactivation of 3-MeSO2-DDE. The thesis forms a basis for further investigations in the human adrenocortical cell line H295R and provides new knowledge of importance for toxicological risk assessment of 3-MeSO2-DDE.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 331
Keyword
Toxicology, 3-methylsulphonyl-DDE, o, p'-DDD, CYP11B1, adrenal cortex, tissue-specific toxicity, bioactivation, kinetics, Y-1 cells, Toxikologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-8180 (URN)978-91-554-6950-4 (ISBN)
Public defence
2007-09-29, Lindahlsalen, Evolutionsbiologiskt centrum (EBC), Norbyvägen 18 A, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2007-09-06 Created: 2007-09-06 Last updated: 2011-01-20Bibliographically approved

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Asp, VendelaBergström, UlrikaBrandt, Ingvar

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