Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.
The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.
- MeSH
- adukty DNA metabolismus MeSH
- DNA nádorová metabolismus MeSH
- karcinogeneze * chemicky indukované metabolismus MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kyseliny aristolochové toxicita MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
The plant extract aristolochic acid (AA), containing aristolochic acid I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy and Balkan endemic nephropathy, unique renal diseases associated with upper urothelial cancer. Differences in the metabolic activation and detoxification of AAI and AAII and their effects on the metabolism of AAI/AAII mixture in the plant extract might be of great importance for an individual's susceptibility in the development of AA-mediated nephropathies and malignancies. Here, we investigated in vivo metabolism of AAI and AAII after ip administration to Wistar rats as individual compounds and as AAI/AAII mixture using high performance liquid chromatography/electrospray ionization mass spectrometry. Experimental findings were supported by theoretical calculations using density functional theory. We found that exposure to AAI/AAII mixture affected the generation of their oxidative and reductive metabolites formed during Phase I biotransformation and excreted in rat urine. Several Phase II metabolites of AAI and AAII found in the urine of exposed rats were also analyzed. Our results indicate that AAI is more efficiently metabolized in rats in vivo than AAII. Whereas AAI is predominantly oxidized during in vivo metabolism, its reduction is the minor metabolic pathway. In contrast, AAII is mainly metabolized by reduction. The oxidative reaction only occurs if aristolactam II, the major reductive metabolite of AAII, is enzymatically hydroxylated, forming aristolactam Ia. In AAI/AAII mixture, the metabolism of AAI and AAII is influenced by the presence of both AAs. For instance, the reductive metabolism of AAI is increased in the presence of AAII while the presence of AAI decreased the reductive metabolism of AAII. These results suggest that increased bioactivation of AAI in the presence of AAII also leads to increased AAI genotoxicity, which may critically impact AAI-mediated carcinogenesis. Future studies are needed to explain the underlying mechanism(s) for this phenomenon.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- injekce intraperitoneální MeSH
- krysa rodu rattus MeSH
- kyseliny aristolochové aplikace a dávkování metabolismus moč MeSH
- potkani Wistar MeSH
- teorie funkcionálu hustoty MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Benzo[a]pyrene (BaP) is an environmental pollutant that, based on evidence largely from in vitro studies, exerts its genotoxic effects after metabolic activation by cytochrome P450s. In the present study, Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice have been used to study the role of P450s in the metabolic activation of BaP in vivo. In HRN mice, cytochrome P450 oxidoreductase (POR), the electron donor to P450, is deleted specifically in hepatocytes. In HBRN mice the microsomal haemoprotein cytochrome b 5 , which can also act as an electron donor from cytochrome b 5 reductase to P450s, is also deleted in the liver. Wild-type (WT), HRN and HBRN mice were treated by i.p. injection with 125 mg/kg body weight BaP for 24 h. Hepatic microsomal fractions were isolated from BaP-treated and untreated mice. In vitro incubations carried out with BaP-pretreated microsomal fractions, BaP and DNA resulted in significantly higher BaP-DNA adduct formation with WT microsomal fractions compared to those from HRN or HBRN mice. Adduct formation (i.e. 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP [dG-N2-BPDE]) correlated with observed CYP1A activity and metabolite formation (i.e. BaP-7,8-dihydrodiol) when NADPH or NADH was used as enzymatic cofactors. BaP-DNA adduct levels (i.e. dG-N2-BPDE) in vivo were significantly higher (~ sevenfold) in liver of HRN mice than WT mice while no significant difference in adduct formation was observed in liver between HBRN and WT mice. Our results demonstrate that POR and cytochrome b 5 both modulate P450-mediated activation of BaP in vitro. However, hepatic P450 enzymes in vivo appear to be more important for BaP detoxification than its activation.
- MeSH
- adukty DNA metabolismus MeSH
- benzopyren metabolismus MeSH
- cytochrom-B(5)-reduktasa metabolismus MeSH
- hepatocyty enzymologie MeSH
- jaterní mikrozomy enzymologie MeSH
- myši knockoutované MeSH
- myši MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(-/-) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(-/-) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(-/-) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation.
- MeSH
- adukty DNA účinky léků genetika MeSH
- arylsulfotransferasa genetika MeSH
- benz(a)anthraceny toxicita MeSH
- cytosol účinky léků metabolismus MeSH
- játra účinky léků metabolismus MeSH
- karcinogeny toxicita MeSH
- kyseliny aristolochové toxicita MeSH
- ledviny účinky léků metabolismus MeSH
- lidé MeSH
- multigenová rodina MeSH
- myši knockoutované MeSH
- myši transgenní MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: The term "endocrine disruptor" (ED) is used for compounds that mimic or antagonize the effects of endogenous hormones. Synthetic estrogen 17α-ethinylestradiol (EE2) and a human carcinogen benzo[a]pyrene (BaP) are assigned as exogenous endocrine disruptors and an estrogenic hormone estradiol is a natural endogenous disruptor. Here, the potency of these three disruptors administered to rats individually and in combination to induce expression of cytochrome P450 (CYP) enzymes involved in their own metabolism (CYP1A1, 2C and 3A) in vivo was investigated. METHODS: Changes in CYP protein expression after exposure of rats to BaP, EE2 or estradiol were analyzed by Western blotting. Using the HPLC method, CYP1A1, 2C and 3A specific activities in hepatic microsomes isolated from exposed rats were analyzed. RESULTS: Whereas exposure to BaP induces expression of CYP1A1 protein and its marker activity (Sudan I oxidation) in liver, kidney and lung of rats, no significant induction of this CYP and its enzyme activity was produced by EE2 and estradiol. Treatment of BaP in combination with EE2 and/or estradiol decreased the BaP-mediated CYP1A1 induction in liver of exposed rats. BaP also induces CYP2C11 protein in rat liver and kidney, but does not increase its enzyme activity measured as testosterone 16α-hydroxylation. The enzyme activity of another enzyme of the 2C subfamily, CYP2C6, diclofenac 4'-hydroxylation, is even decreased by BaP. The CYP2C11 protein expression and/or its activity are also increased in liver of rats treated with EE2 and estradiol, but its expression is significantly decreased in lung. The CYP2C6 activity is also elevated by treatment of rats with EE2 and estradiol administered individually as well as in their combination. Whereas only a slight increase in CYP3A protein expression was found by BaP in rat liver, its enzyme activity, testosterone 6β-hydroxyalation, increased significantly in this organ. In contrast, no effect or even a decrease in CYP3A expression and its enzyme activity was produced by EE2 and estradiol in rats exposed to these compounds.
- MeSH
- benzopyren farmakologie MeSH
- endokrinní disruptory farmakologie MeSH
- estradiol farmakologie MeSH
- estrogeny farmakologie MeSH
- ethinylestradiol farmakologie MeSH
- jaterní mikrozomy metabolismus MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: 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.
- MeSH
- adukty DNA metabolismus MeSH
- aromatické hydroxylasy genetika metabolismus MeSH
- cytochrom P-450 CYP1A1 MeSH
- cytochrom P-450 CYP1A2 MeSH
- cytochrom P450 CYP1B1 MeSH
- katalytická doména genetika MeSH
- katalýza MeSH
- kyseliny aristolochové metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- mutageneze cílená MeSH
- oxidace-redukce MeSH
- rekombinantní proteiny MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.
- MeSH
- adukty DNA antagonisté a inhibitory biosyntéza MeSH
- cytochrom P-450 CYP1A1 biosyntéza MeSH
- cytochrom P-450 CYP1A2 biosyntéza MeSH
- enzymová indukce účinky léků fyziologie MeSH
- karcinogeny toxicita MeSH
- krysa rodu rattus MeSH
- kyseliny aristolochové toxicita MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aristolochic acid I (AAI) is a plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is detoxified by cytochrome P450 (CYP)-mediated O-demethylation to 8-hydroxyaristolochic acid I (aristolochic acid Ia, AAIa). We previously investigated the efficiencies of human and rat CYPs in the presence of two other components of the mixed-functions-oxidase system, NADPH:CYP oxidoreductase and cytochrome b₅, to oxidize AAI. Human and rat CYP1A are the major enzymes oxidizing AAI. Other CYPs such as CYP2C, 3A4, 2D6, 2E1, and 1B1, also form AAIa, but with much lower efficiency than CYP1A. Based on velocities of AAIa formation by examined CYPs and their expression levels in human and rat livers, here we determined the contributions of individual CYPs to AAI oxidation in these organs. Human CYP1A2 followed by CYP2C9, 3A4 and 1A1 were the major enzymes contributing to AAI oxidation in human liver, while CYP2C and 1A were most important in rat liver. We employed flexible in silico docking methods to explain the differences in AAI oxidation in the liver by human CYP1A1, 1A2, 2C9, and 3A4, the enzymes that all O-demethylate AAI, but with different effectiveness. We found that the binding orientations of the methoxy group of AAI in binding centers of the CYP enzymes and the energies of AAI binding to the CYP active sites dictate the efficiency of AAI oxidation. Our results indicate that utilization of experimental and theoretical methods is an appropriate study design to examine the CYP-catalyzed reaction mechanisms of AAI oxidation and contributions of human hepatic CYPs to this metabolism.
- MeSH
- inhibitory cytochromu P450 farmakologie MeSH
- jaterní mikrozomy účinky léků metabolismus MeSH
- játra účinky léků metabolismus MeSH
- katalytická doména MeSH
- katalýza MeSH
- krysa rodu rattus MeSH
- kyseliny aristolochové škodlivé účinky chemie metabolismus MeSH
- lidé MeSH
- metabolická aktivace MeSH
- metabolická inaktivace MeSH
- metylace účinky léků MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- nemoci ledvin etiologie metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- systém (enzymů) cytochromů P-450 chemie metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
