Defining in vivo dose-response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach.

Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.

DNA Adducts Formed by Aristolochic Acid Are Unique Biomarkers of Exposure and Explain the Initiation Phase of Upper Urothelial Cancer.

Aristolochic acid (AA) is a plant alkaloid that causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases frequently associated with upper urothelial cancer (UUC). This review summarizes the significance of AA-derived DNA adducts in the aetiology of UUC leading to specific A:T to T:A transversion mutations (mutational signature) in AAN/BEN-associated tumours, which are otherwise rare in individuals with UCC not exposed to AA. Therefore, such DNA damage produced by AA-DNA adducts is one rare example of the direct association of exposure and cancer development (UUC) in humans, confirming that the covalent binding of carcinogens to DNA is causally related to tumourigenesis. Although aristolochic acid I (AAI), the major component of the natural plant extract AA, might directly cause interstitial nephropathy, enzymatic activation of AAI to reactive intermediates capable of binding to DNA is a necessary step leading to the formation of AA-DNA adducts and subsequently AA-induced malignant transformation. Therefore, AA-DNA adducts can not only be utilized as biomarkers for the assessment of AA exposure and markers of AA-induced UUC, but also be used for the mechanistic evaluation of its enzymatic activation and detoxification. Differences in AA metabolism might be one of the reasons for an individual's susceptibility in the multi-step process of AA carcinogenesis and studying associations between activities and/or polymorphisms of the enzymes metabolising AA is an important determinant to identify individuals having a high risk of developing AA-mediated UUC.

Metabolic activation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/-) and Trp53(-/-) mice.

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)-mediated biotransformation. The carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with a single oral dose of 50 mg/kg body weight PhIP. N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) levels in DNA, measured by liquid chromatography-tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(-/-) mice compared to Trp53(+/+) mice. Lower PhIP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin-O-demethylase activity) in these animals. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/-) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2-naphthylsulfate formation from 2-naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue-dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP-DNA adduct formation.

7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1.

Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H: quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using (32)P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.

DNA adducts as link between in vitro and in vivo carcinogenicity - A case study with benzo[a]pyrene.

To reduce the need for animal tests, in vitro assays are often used as alternative methods. To derive toxic doses for higher tier organisms from in vitro assay results, quantitative in vitro-in vivo extrapolation (qIVIVE) based on physiological-based toxicokinetic (PBTK) models is typically the preferred approach. Such PBTK models require many input parameters to address the route from dose to target site concentration. However, respective data is very often not available. Hence, our aim is to call attention to an alternative way to build a link between animal (in vivo) and cell-derived (in vitro) toxicity data. To this end, we selected the carcinogenic chemical benzo[a]pyrene (BaP) for our study. Our approach relates both in vitro assay and in vivo data to a main intermediate marker structure for carcinogenicity on the subcellular level - the BaP-DNA adduct BaP-7,8-dihydrodiol-9,10-epoxide-deoxyguanosine. Thus, BaP dose is directly linked to a measure of the toxicity-initiating event. We used Syrian hamster embryo (SHE) and Balb/c 3T3 cell transformation assay as in vitro data and compared these data to outcomes of in vivo carcinogenicity tests in rodents. In vitro and in vivo DNA adduct levels range within three orders of magnitude. Especially metabolic saturation at higher doses and interspecies variabilities are identified and critically discussed as possible sources of errors in our simplified approach. Finally, our study points out possible routes to overcome limitations of the envisaged approach in order to allow for a reliable qIVIVE in the future.

In vitro and in silico study on consequences of combined exposure to the food-borne alkenylbenzenes estragole and safrole.

Potential consequences of combined exposure to the selected food-borne alkenylbenzenes safrole and estragole or their proximate carcinogenic 1'-hydroxy metabolites were evaluated in vitro and in silico. HepG2 cells were exposed to 1'-hydroxyestragole and 1'-hydroxysafrole individually or in equipotent combination subsequently detecting cytotoxicity and DNA adduct formation. Results indicate that concentration addition adequately describes the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts. Furthermore, physiologically based kinetic modeling revealed that at normal dietary intake the concentration of the parent compounds and their 1'-hydroxymetabolites remain substantially below the Km values for the respective bioactivation and detoxification reactions providing further support for the fact that the simultaneous presence of the two carcinogens or of their proximate carcinogenic 1'-hydroxy metabolites may not affect their DNA adduct formation. Overall, these results point at the absence of interactions upon combined exposure to selected food-borne alkenylbenzenes at realistic dietary levels of intake.

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

BACKGROUND/AIM: Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. MATERIALS AND METHODS: The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. RESULTS: DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. CONCLUSION: DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Development of novel and safer anti-breast cancer agents, SS1020 and SS5020, based on a fundamental carcinogenic research.

Tamoxifen (TAM) has been prescribed worldwide to patients with and women at high-risk of breast cancer. However, long-term use of TAM increases the incidence of endometrial cancer. The carcinogenic mechanisms of TAM have been extensively investigated. TAM is hydroxylated and sulfonated at α-carbon to form α-hydroxytamoxifen-O-sulfonate. This metabolite readily reacts with genomic DNA, particularly with 2'-deoxyguanosine, leading to DNA replication error. TAM also exerts estrogenic activity at endometrial tissue to induce endometrial hyperplasia. Therefore, our efforts focused on the development of novel and safer anti-estrogens to diminish carcinogenic potential of TAM based on chemical modifications. In this review, we describe a crucial idea of our drug design and introduce our compounds SS1020 and SS5020, possessing high effectiveness, and no genotoxic and estrogenic activities.

Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts.

We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 µM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.

Protective role of cytochrome P450 1A1 (CYP1A1) against benzo[a]pyrene-induced toxicity in mouse aorta.

Benzo[a]pyrene (BaP) is an environmental pollutant produced by combustive processes, such as cigarette smoke and coke ovens, and is implicated in the pathogenesis of atherosclerosis. Cytochrome P450 1A1 (CYP1A1) plays a role in both metabolic activation and detoxication of BaP in a context-dependent manner. The role of CYP1A1 in BaP-induced toxicity in aorta remains unknown. First, we fed Apoe⁻/⁻ mice an atherogenic diet plus BaP and found that oral BaP-enhanced atherosclerosis is associated with increased reactive oxygen species (ROS) and inflammatory markers, such as plasma tumor necrosis factor levels and aortic mRNA expression of vascular endothelial growth factor A (Vegfa). We next examined the effect of an atherogenic diet plus BaP on ROS and inflammatory markers in Cyp1a1⁻/⁻ mice. Although this treatment was not sufficient to induce atherosclerotic lesions in Cyp1a1⁻/⁻ mice, plasma antioxidant levels were decreased in Cyp1a1⁻/⁻ mice even in the absence of BaP treatment. The atherogenic diet plus BaP effectively elevated plasma ROS levels and expression of atherosclerosis-related genes, specifically Vegfa, in Cyp1a1⁻/⁻ mice compared with wild-type mice. BaP treatment increased Vegfa mRNA levels in mouse embryonic fibroblasts from Cyp1a1⁻/⁻ mice but not from wild-type mice. BaP-induced DNA adduct formation was increased in the aorta of Cyp1a1⁻/⁻ mice, but not wild-type or Apoe⁻/⁻ mice, and the atherogenic diet decreased BaP-induced DNA adducts in Cyp1a1⁻/⁻ mice compared with mice on a control diet. These data suggest that ROS production contributes to BaP-exacerbated atherosclerosis and that CYP1A1 plays a protective role against oral BaP toxicity in aorta.