Elsevier

Toxicology Letters

Volume 212, Issue 2, 20 July 2012, Pages 97-105
Toxicology Letters

Role of mammary epithelial and stromal P450 enzymes in the clearance and metabolic activation of 7,12-dimethylbenz(a)anthracene in mice

https://doi.org/10.1016/j.toxlet.2012.05.005Get rights and content

Abstract

Microsomal cytochrome P450 (P450) enzymes, which are important in the metabolism of carcinogens, are expressed in both epithelial and stromal cells in the mammary gland. The aim of this study was to investigate the roles of mammary epithelial P450 enzymes in the bioactivation and disposition of 7,12-dimethylbenz(a)anthracene (DMBA), a breast carcinogen, in the mammary gland. A new mouse model (named MEpi-Cpr-null) was produced, wherein P450 activities in the mammary epithelial cells are suppressed through tissue-specific deletion of the gene for P450 reductase (Cpr), an enzyme required for the activities of all microsomal P450 enzymes. Comparisons between wild-type and MEpi-Cpr-null mice showed that the tissue-specific deletion of Cpr in the mammary epithelial cells was accompanied by significant increases in the levels of DMBA and DMBA–DNA adduct in the mammary gland following a single intraperitoneal injection of DMBA at 50 mg/kg. Immunohistochemical and immunoblot analysis further revealed greater induction of CYP1B1 expression by the DMBA treatment in the mammary stroma of the MEpi-Cpr-null mice than in that of the WT mice. These findings not only demonstrate that the epithelial P450 enzymes play important roles in the clearance of DMBA, but also suggest that P450 enzymes in both mammary epithelial and stromal cells contribute to carcinogen-mediated DNA damage.

Highlights

► A new mouse model having suppressed P450 activities in the mammary epithelial cells. ► Increased DMBA level in the mammary gland following DMBA treatment. ► Increased DMBA–DNA adduct level in the mammary gland following DMBA treatment.

Introduction

The etiology of breast cancer is largely unknown. Exposure to environmental carcinogens has been proposed as a possible contributing factor, although experimental proof or epidemiological evidence for associating breast cancer with exposures to a particular environmental compound has yet to be obtained. Most chemicals require metabolic activation to become ultimate carcinogens. Microsomal cytochrome P450 (P450 or CYP) monooxygenases play essential roles in the metabolism of chemical carcinogens (Guengerich, 1988).

Although the liver is the major site for metabolic disposition and activation of chemical carcinogens, some fatty extrahepatic tissues, including the mammary gland, can accumulate hydrophobic compounds, such as polycyclic aromatic hydrocarbons (PAH), thus increasing the potential importance of local metabolic activation in PAH-induced mammary carcinogenesis. The ability of the mammary gland to metabolically activate procarcinogens has been demonstrated by studies in which the chemicals were directly injected into mammary gland of rodents, resulting in localized DNA adduct formation (Arif et al., 1997, Todorovic et al., 1997). Aromatic DNA adducts have been detected in human breast tissue (Perera et al., 1995, Li et al., 1996) and confirmed to be related to PAH exposure; their presence was also associated with the genetic polymorphisms of CYP1A1 (Li et al., 2002), a major P450 isoform for the metabolism of PAH carcinogens. Many P450 isoforms have been detected in rat breast tissue through immunoblot analysis, including CYP1A1, 1A2, 2A, 2B, 2D4, 3A, 4A, 2E1, and 19 (Hellmold et al., 1995, Hellmold et al., 1998). The expression of CYP1A1, 1B1, 2C, 2D6, 2E1, and 3A4/5 mRNAs was also detected in the human breast tissue (Williams and Phillips, 2000). The expression of CYP2A5, CYP2B, CYP3A, and CYP19 has also been detected in the mouse mammary gland by immunoblot analysis (Gu et al., unpublished results).

DMBA (7,12-dimethylbenz(a)anthracene), is one of the most potent mammary carcinogens in animals, including mice (Median, 1982). DMBA requires multiple steps of metabolic activation; the resulting ultimate carcinogens are unstable and short lived. Stable DNA adducts are detectable in rodent mammary tissue following oral treatment with DMBA (Izzotti et al., 1999, Kleiner et al., 2001). Both CYP1A1 and CYP1B1 are major P450 isoforms for metabolic activation of PAH carcinogens, including DMBA, based on in vitro and in vivo metabolism studies (Parkinson and Ogilvie, 2008, Kleiner et al., 2004). CYP1A1 and CYP1B1 display stereospecific metabolism of DMBA, with CYP1A1 producing the anti-diolepoxides and CYP1B1 producing the syn-isomer.

The importance of potential interplays between the epithelial and stromal cell populations in breast development and carcinogenesis has been increasingly recognized in recent years (Wiseman and Werb, 2002). However, although metabolic activation in the mammary gland is believed to play a major role in chemical carcinogenesis in the breast tissue, the respective roles of the epithelial and stromal P450s in chemical carcinogenesis are largely unknown. In that connection, we have been developing in vivo models for determining tissue-specific contributions to chemical toxicity, including the relative contributions of the epithelial and stromal P450 enzymes to chemical carcinogenesis in the breast tissue.

The NADPH-P450 reductase (CPR) is the obligate redox partner for microsomal P450 enzymes (Black and Coon, 1987); deletion of the Cpr gene results in the inactivation of all microsomal P450 enzymes in targeted cells or tissues (Gu et al., 2003). Germline disruption of the mouse Cpr gene led to a spectrum of embryonic defects and mid-gestational lethality, indicating that CPR is essential for early embryonic development (Shen et al., 2002). Through crossbreeding between the Cpr–lox mouse (Wu et al., 2003) and various Cre transgenic mice, several tissue-specific Cpr-null mouse models have been produced, including the liver-specific Cpr-null mouse (Gu et al., 2003), the lung-specific Cpr-null mouse (Weng et al., 2007), the cardiomyocyte-specific Cpr-null mouse (Fang et al., 2008a), the intestinal epithelium-specific Cpr-null mouse (Zhang et al., 2009), and the brain neuron-specific Cpr-null mouse (Conroy et al., 2010). Studies on these tissue-specific Cpr-null models have yielded direct evidence for the roles of P450 enzymes in the metabolic activation or disposition of various drugs and toxicants in the targeted tissues and organs.

The aim of this study was to develop a mammary epithelium-specific Cpr-null mouse, and to apply this model to determine the role of mammary P450 enzymes in the metabolic activation of PAH carcinogens (such as DMBA). Here, we report the successful generation of a mammary epithelium-specific Cpr-null (MEpi-Cpr-null) mouse model, produced through crossbreeding between the Cpr–lox mouse and the MMTV-Cre mouse; the latter is a well-characterized Cre transgenic mouse, widely used in many studies for mammary epithelium-specific gene deletion (Wagner et al., 2001, Wagner et al., 2003, Cui et al., 2002, Loladze et al., 2006, Feng et al., 2007). We confirmed specific deletion of the Cpr gene in mammary epithelial cells, through immunohistochemical analysis. We then compared tissue levels of DMBA and DMBA–DNA adducts in DMBA-treated WT and MEpi-Cpr-null mice. We further examined expression of CYP1A1 and CYP1B1, two P450 enzymes possibly involved in DMBA metabolism in the mammary gland, through immunohistochemical and immunoblot analyses. We believed that our studies on the MEpi-Cpr-null mouse have yielded the first direct evidence for the specific role of mammary epithelial (vs. stromal) P450 enzymes in the metabolic disposition and activation of a PAH carcinogen.

Section snippets

Generation of the MEpi-Cpr-null mice

The MMTV-Cre transgenic mouse (on a mixed B6/129 background) was obtained from Jackson Laboratory (Bar Harbor, ME) (Wagner et al., 2001). The Cpr–lox mouse [Cpr(lox/lox)]; congenic on B6 background) (Wu et al., 2003), was available at the Wadsworth Center. MMTV-Cre hemizygous transgenic mice were first crossed with Cpr(lox/lox) mice to generate MMTV-Cre(±)Cpr(lox/−) mice, which were crossed again with Cpr(lox/lox) mice, producing MMTV-Cre(±)Cpr(lox/lox) mice (designated MEpi-Cpr-null) and

General characterization of the MEpi-Cpr-null mice

The MEpi-Cpr-null mice were viable, fertile, and normal in size and body weight, and exhibit no obvious physical or behavioral abnormities, compared to WT littermates. There was no embryonic lethality in the MEpi-Cpr-null mice, based on analyses of genotype distribution in pups derived from crossbreeding between MMTV-Cre(±)/Cpr(lox/−) and MMTV-Cre(−/−)/Cpr(lox/lox) mice (data not shown). Histological examination of the mammary glands of the MEpi-Cpr-null mice did not reveal any structural

Discussion and conclusion

In recent years, with the availability of tissue-selective Cpr-null mouse models, it has become possible to directly determine the in vivo contributions of P450 enzymes in a given organ or tissue to the disposition and toxicity of numerous xenobiotics. Evidence obtained so far indicated that extrahepatic P450 enzymes play important roles in the in situ metabolic activation and disposition of various drugs and toxicants (e.g., Weng et al., 2007, Fang et al., 2008b, Xiao et al., 2008, Zhang et

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We also gratefully acknowledge the use of the Biochemistry, Advanced Light Microscopy, and Histopathology Core facilities of the Wadsworth Center. This work was supported in part by funds from MOST2010DFB30270/2011CB964800 and Tianjin 09ZCZDSF03800 (to T.C.), and NIH grants ES009132, ES019869, and HL087174 (to B.M), CA092596 (to X.D.), and ES018884 (to J.G.).

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