Sudha R. Kondraganti

Polycyclic aromatic hydrocarbon-inducible DNA adducts: Evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3‐methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR‐null animals are less susceptible to PAH‐induced DNA adduct formation than wild‐type animals. Wild‐type [AHR (+/+)] mice or mice lacking the gene for the AHR were treated with a single dose (100 μmol/kg) of BP or MC, and hepatic DNA adducts were analyzed by 32P‐postlabeling. BP induced multiple hepatic DNA adducts in wild‐type as well as AHR‐null animals, suggesting the existence of AHR‐independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHR‐null animals exposed to MC, although the major MC‐DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7‐ethoxyresorufin O‐deethylase (EROD) (CYP1A1) and 7‐methoxyresorufin O‐demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild‐type, but not, in AHR‐null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild‐type animals. In conclusion, these results demonstrate the existence of AHR‐ and CYP1A1‐independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH‐mediated carcinogenesis.

Persistent Induction of Hepatic and Pulmonary Phase II Enzymes by 3-Methylcholanthrene in Rats

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.

Differential regulation of expression of hepatic and pulmonary cytochrome P4501A enzymes by 3-methylcholanthrene in mice lacking the CYP1A2 gene

The cytochrome P4501A enzymes play important roles in carcinogen metabolism. We reported previously that 3-methylcholanthrene (MC) elicits a persistent induction of hepatic, pulmonary, and mammary microsomal cytochrome P450 (P450) 1A enzymes for several weeks after MC withdrawal. In this study, we tested the hypothesis that CYP1A2, a liver-specific P450 isozyme, plays an important role in the mechanisms governing persistent CYP1A1 induction by MC in liver but not in extra-hepatic tissues such as lung, which is devoid of endogenous CYP1A2. Administration of wild-type (WT) or CYP1A2-null mice with MC (100 μmol/kg i.p.) once daily for 4 days caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein contents, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not CYP1A2-null animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for 15 days in both the genotypes. Since MC is almost completely eliminated by day 15, we hypothesize that CYP1A2 contributes to the up-regulation of CYP1A1 in liver, but not lung, by a novel mechanism, presumably involving a CYP1A2-dependent persistent metabolite. The studies demonstrate tissue-specific differences in the regulation of CYP1A by MC, a phenomenon that may have implications for human carcinogenesis caused by environmental chemicals.

Abstract 4427: Role of cytochrome P450 (CYP)1A2 in the sustained induction of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1)

Humans are constantly exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs) through cigarette smoke, diesel exhausts, charcoal-broiled meats, etc. Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC ithdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in Cyp1a2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 nduction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this henomenon. Hepa-1 cells were treated ith the MC (2.5 µM), or dimethylsulfoxide (DMSO) as control, and at selected ime points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. We fund that MC markedly and persistently induced CYP1A1 promoter activity, transcription, apoprotein expression, and the CYP1A1 associated ethoxyresorufin O-deethylase (EROD) activities for up to 5 days. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter ctivity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly ttenuated by Cyp1a2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans. (Supported in part by 2R01 ES009132.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4427. doi:1538-7445.AM2012-4427

Abstract 825: Molecular mechanisms of regulation of cytochrome P4501A enzymes by 3-methylcholanthrene (MC) in mice in vivo

3-Methylcholanthrene (MC) is one of the most potent polycyclic aromatic hydrocarbons (PAHs) present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP)1A enzymes play key roles in the activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by MC in vivo. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 in vivo by sustained transcriptional activation of the CYP1A1 promoter. Thirty two C57B6 (WT) mice were divided into two groups. Group I was treated with vehicle corn oil (CO) (8ml/kg) and group II was treated with a single dose of MC (100 μmol/kg), i.p. Four animals from each group were sacrificed at 6, 12, 24, and 48 h after MC withdrawal. The mRNA levels, protein content and enzyme activities of CYP1A1 were determined by real-time PCR, Western blotting, and fluorimetry, respectively at different time points. In addition, the binding of MC-AHR-AHR nuclear translocator (ARNT) to the AHREs on the CYP1A1 promoter region were determined by chromatin immunoprecipitation (ChIP) assay. The translocation of AHR was also analyzed by immunofluorescence. The ChIP experiments indicated that transcriptional activation of CYP1A1 was most pronounced at 6 h, followed by 12 h, but declined at later time points. On the other hand, the expression of CYP1A1 at the mRNA, protein and enzyme levels persisted for up to 48 h both in lung and liver tissues. These results suggest that transcriptional activation of CYP1A1 at 6-12 h is sufficient to result in sustained induction of CYP1A mRNA and protein expression for up to 48 h. Isolation of the immunoprecipitated promoter fragment (containing the xenobiotic response elements), which binds to the MC-AHR complex, followed by 32P-postlabeling revealed the formation of MC-DNA adducts, suggesting that DNA adducts are sequence-specific and target the CYP1A1 promoter. Our results suggest that DNA adducts might play a role in regulation of CYP1A1 by MC, a phenomenon that may be of relevance to PAH-mediated carcinogenesis. Citation Format: Bhagavatula Moorthy, Jiang Weiwu, Lihua Wang, Chun Chu, Sudha R. Kondraganti, Paramahamsa Maturu. Molecular mechanisms of regulation of cytochrome P4501A enzymes by 3-methylcholanthrene (MC) in mice in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 825. doi:10.1158/1538-7445.AM2015-825

Abstract 1587: Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnHumans are frequently exposed to polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP)1A and 1B1 enzymes are known to play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to DNA-binding metabolites, which in turn mediate carcinogenesis in target organs such as lung, breast, and skin. In this investigation, we tested the hypothesis that mice lacking the gene for Cyp1b1 will show attenuation of MC-DNA adduct formation, compared to WT mice. Eight week-old female wild type (WT) (C57BL/6J) mice or mice lacking the gene for Cyp1b1 were treated with MC (100 µmol/kg), once daily for 2 days, and animals sacrificed 1 day after the last treatment. DNA adducts from liver and lung genomic DNA were analyzed by the 32P-postabeling assay. Control animals received only the vehicle corn oil (CO). In WT mice, CO-treated group did not show any adducts, whereas MC-treated animals showed 11 adduct spots in the lung. In Cyp1b1-null mice, although there were no qualitative differences in the pulmonary adduct pattern, the levels of each adduct was markedly suppressed (25-90%), with total levels of adducts being about 15% of those formed in WT mice. In liver of WT as well as Cyp1b1-null mice, MC induced formation of 11 adducts, and the adduct pattern was similar to that of lung, but the total levels of adducts in WT mice were about a third of those seen in the lungs of the corresponding animals. In Cyp1b1-null mice, MC adducts were significantly decreased compared to WT mice, but the extent of attenuation in liver was lesser than that in lung. These results suggest that CYP1B1 plays a major role in formation of DNA adducts from MC. Because DNA adducts are pre-mutagenic lesions, our results are highly relevant to PAH-mediated pulmonary carcinogenesis. Our data suggest that CYP1B1 could be a molecular target for developing preventive and therapeutic strategies against lung cancer in humans.nnCitation Format: Bhagavatula Moorthy, Guodong Zhou, Lihua Wang, Sudha R. Kondraganti, Weiwu Jiang. Mechanistic role of cytochrome P4501B1 in DNA adduct formation by 3-methylcholanthrene in mice: Implications for lung cancer in humans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1587. doi:10.1158/1538-7445.AM2014-1587

Abstract 769: Regulation of drug metabolizing enzyme and transporter gene expression by dietary and chemopreventive phytochemicals in breast cancer

Breast cancer is a leading cause of cancer-related death among American women. The etiology of majority of breast cancers is multifactorial, of which 95% is environmental and hormonal. Polycyclic aromatic hydrocarbons (PAHs) are commonly found in our environment and appear to be carcinogenic by genotoxic mechanisms. The taxanes are the most unique and successful chemotherapeutic agents used for the treatment of breast and ovarian cancer. However, multi-drug resistance (MDR) is one of the primary obstacles for successful chemotherapy. Drug metabolizing enzymes (DMEs) and transporters play important roles in modulating drug absorption, distribution, metabolism, and elimination. The regulation of gene expression of DMEs and transporters has potential impact on drug interactions of many therapeutic agents. P-glycoprotein (P-gp), Pregnane X receptor (PXR), and CYP3A are associated with MDR in breast cancer chemotherapy. Taxanes induce CYP3A by activating PXR-mediated transcription and enhance P-gp mediated drug clearance in human mammary tumor cells. Recent studies involving drug-phytochemical interactions, in addition to interactions among dietary micronutrients, indicate possibilities for improved therapeutic strategies. It is estimated that roughly 50% of cancer patients use some kind of dietary supplements. In the present study, we evaluated the ability of dietary and chemopreventive phytochemicals (curcumin and sulforaphane) on reversal of MDR induced by taxanes (paclitaxel and docetaxel). We also assessed the effects of dietary phytochemicals and taxanes, alone or in combination, on the gene expression of various DMEs, transporters, and receptors in female C57BL/6J mice exposed to the environmental PAH, 7, 12-dimethylbenz(a)anthracene (DMBA). The dietary phytochemicals suppressed the hepatic and mammary gene expression of Phase 1 (CYP1A1, 1B1) DMEs and elevated the levels of Phase 2 (NQO1) DMEs. The hepatic gene expression of CYP3A11, PXR, aryl hydrocarbon receptor and mammary gene expression of P-gp were induced in female mice exposed to DMBA and chemotherapeutic drugs (paclitaxel and docetaxel). Administration of curcumin and sulforaphane inhibited the induced gene expression levels significantly. Treatment of mice with a combination of curcumin and sulforaphane was more effective compared to treatment with either curcumin or sulforaphane alone. The differential regulation of gene expression levels reveal the potential of phytochemicals to reverse MDR in breast cancer chemotherapy. These studies will help in development of effective pharmacotherapeutic based strategies for preventing and treating human breast cancer. The studies will also lead to the development of novel combination drugs that can be used for successful chemotherapy of breast cancer in women. Citation Format: Sudha Kondraganti, Lihua Wang, Weiwu Jiang, Bhagavatula Moorthy. Regulation of drug metabolizing enzyme and transporter gene expression by dietary and chemopreventive phytochemicals in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 769. doi:10.1158/1538-7445.AM2014-769