Induction and localization of cytochrome P450 1B1 (CYP1B1) protein in the livers of TCDD-treated rats: Detection using polyclonal antibodies raised to histidine-tagged fusion proteins produced and purified from bacteria

Nigel J. Walker, Frances G. Crofts, Ying Li, Sigurd F. Lax, Carrie L. Hayes, Paul Timothy Strickland, George W. Lucier, Thomas R. Sutter

Research output: Contribution to journalArticle

Abstract

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague Dawley rats following either acute (single dose of 25 μg TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.

Original languageEnglish (US)
Pages (from-to)395-402
Number of pages8
JournalCarcinogenesis
Volume19
Issue number3
DOIs
StatePublished - Mar 1998

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Histidine
Cytochrome P-450 Enzyme System
Cytochrome P-450 CYP1A1
Bacteria
Antibodies
Liver
Cytochrome P-450 CYP1A2
Proteins
His-His-His-His-His-His
Polychlorinated Dibenzodioxins
Escherichia coli Proteins
Cytochromes
Isoenzymes
Sprague Dawley Rats
Hepatocytes
Rodentia
Rabbits
Peptides

ASJC Scopus subject areas

  • Cancer Research

Cite this

Induction and localization of cytochrome P450 1B1 (CYP1B1) protein in the livers of TCDD-treated rats : Detection using polyclonal antibodies raised to histidine-tagged fusion proteins produced and purified from bacteria. / Walker, Nigel J.; Crofts, Frances G.; Li, Ying; Lax, Sigurd F.; Hayes, Carrie L.; Strickland, Paul Timothy; Lucier, George W.; Sutter, Thomas R.

In: Carcinogenesis, Vol. 19, No. 3, 03.1998, p. 395-402.

Research output: Contribution to journalArticle

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abstract = "Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague Dawley rats following either acute (single dose of 25 μg TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.",
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