Alarmins and Related Molecules in Elective Surgery.

Surgery is associated with alterations of alarmins' and related molecules' levels. The aim of this study was to investigate which biomarkers are most involved in surgery. The studied group consisted of 58 patients with inguinal or umbilical hernia or cholecystolithiasis and 21 healthy controls for compa-rison. We also added seven acute patients with appendicitis, cholecystitis and incarcerated hernia. Serum concentrations of soluble receptor of advanced glycation end-products (sRAGE), extracellular newly identified receptor for advanced glycation end-products binding protein (EN-RAGE), calprotectin, high mobility group box 1 (HMGB1) and interleukin 6 (IL-6) were analysed by ELISA before and after surgery. Preoperative concentrations of calprotectin were significantly decreased while concentrations of sRAGE were significantly increased in patients compared to controls; the concentrations of EN-RAGE and HMGB1 did not differ significantly. IL-6 levels were undetectable in elective patients preoperatively and in controls. Postoperatively, there was a significant increase of EN-RAGE, calprotectin, HMGB1, and IL-6 and a significant decrease of sRAGE compared to preoperative levels. In acute patients, all tested molecules except for sRAGE were significantly increased preoperatively, and sRAGE was significantly decreased. In contrast, after surgery, we could observe a further increase in IL-6; the other biomarkers did not differ significantly. We can conclude that the concentrations of all tested biomarkers are significantly influenced by elective surgery. The postoperative levels of all tested molecules increase except for sRAGE, whose level is significantly decreased after surgery. In acute states, these molecules are already increased, and the influence of surgery is, apart from IL-6, insignificant.

CSF Markers of Oxidative Stress Are Associated with Brain Atrophy and Iron Accumulation in a 2-Year Longitudinal Cohort of Early MS.

In this prospective longitudinal study, we quantified regional brain volume and susceptibility changes during the first two years after the diagnosis of multiple sclerosis (MS) and identified their association with cerebrospinal fluid (CSF) markers at baseline. Seventy patients underwent MRI (T1 and susceptibility weighted images processed to quantitative susceptibility maps, QSM) with neurological examination at the diagnosis and after two years. In CSF obtained at baseline, the levels of oxidative stress, products of lipid peroxidation, and neurofilaments light chain (NfL) were determined. Brain volumetry and QSM were compared with a group of 58 healthy controls. In MS patients, regional atrophy was identified in the striatum, thalamus, and substantia nigra. Magnetic susceptibility increased in the striatum, globus pallidus, and dentate and decreased in the thalamus. Compared to controls, MS patients developed greater atrophy of the thalamus, and a greater increase in susceptibility in the caudate, putamen, globus pallidus and a decrease in the thalamus. Of the multiple calculated correlations, only the decrease in brain parenchymal fraction, total white matter, and thalamic volume in MS patients negatively correlated with increased NfL in CSF. Additionally, negative correlation was found between QSM value in the substantia nigra and peroxiredoxin-2, and QSM value in the dentate and lipid peroxidation levels.

Increased Transferrin Sialylation Predicts Phenoconversion in Isolated REM Sleep Behavior Disorder.

BACKGROUND: Sialic acid-protein interactions are involved in regulating central nervous system immunity; therefore, derangements in sialylation could be involved in neurodegeneration. OBJECTIVES: We evaluate the differences in serum transferrin sialylation in prodromal and early-stage Parkinson's disease (PD), its relation to substantia nigra degeneration, and the risk of phenoconversion to manifest disease. METHODS: Sixty treatment-naive PD patients; 72 polysomnography-confirmed isolated rapid eye movement sleep behavior disorder (iRBD) patients, that is, patients with prodromal synucleinopathy; and 46 healthy volunteers aged ≥45 years and drinking ≤60 standard drinks per month were included. The proportion of serum low-sialylated, carbohydrate-deficient transferrin (CDT) isoforms was assessed using high-performance liquid chromatography, and the values were adjusted for alcohol intake (CDTadj ). Dopamine transporter single-photon emission computed tomography (DaT-SPECT) imaging was performed. In iRBD, phenoconversion risk of DaT-SPECT and CDTadj was evaluated using Cox regression adjusted for age and sex. RESULTS: Median CDTadj was lower in PD (1.1 [interquartile range: 1.0-1.3]%) compared to controls (1.2 [1.1-1.6]%) (P = 0.001). In iRBD, median CDTadj was lower in subjects with abnormal (1.1 [0.9-1.3]%) than normal (1.3 [1.2-1.6]%) DaT-SPECT (P = 0.005). After a median 44-month follow-up, 20% of iRBD patients progressed to a manifest disease. Although iRBD converters and nonconverters did not significantly differ in CDTadj levels (P = 0.189), low CDTadj increased the risk of phenoconversion with hazard ratio 3.2 (P = 0.045) but did not refine the phenoconversion risk associated with abnormal DaT-SPECT yielding hazard ratio 15.8 (P < 0.001). CONCLUSIONS: Decreased serum CDTadj is associated with substantia nigra degeneration in synucleinopathies. iRBD patients with low CDTadj are more likely to phenoconvert to manifest disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Pregnancy-Associated Plasma Protein A2 in Hemodialysis Patients: Significance for Prognosis.

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A) is associated with adverse outcome of long-term hemodialysis patients (HD). The aim of the study was to test whether its homolog pregnancy-associated plasma protein A2 (PAPP-A2) can be detected in serum of HD patients and to define its significance. METHODS: The studied group consisted of 102 long-term HD patients and 25 healthy controls. HD patients were prospectively followed up for five years (2009-2014). PAPP-A2 was measured by surface plasmon resonance biosensor, PAPP-A by time resolved amplified cryptate emission. RESULTS: PAPP-A2, similarly as PAPP-A, was significantly increased in HD patients (median (interquartile range)) PAPP-A2: 6.2 (2.6-10.8) ng/mL, vs. 3.0 (0.7-5.9) ng/mL, p=0.006; PAPP-A: 18.9 (14.3-23.4) mIU/L, vs. 9.5 (8.4-10.5) mIU/L, p<0.001). In HD patients, PAPP-A2 correlated weakly but significantly with PAPP-A (τ=0.193, p=0.004). Unlike PAPP-A, PAPP-A2 was not significant for prognosis of HD patients when tested alone. There was a significant interaction between PAPP-A and PAPP-A2 on the mortality due to infection of HD patients (p=0.008). If PAPP-A was below median, mortality due to infection was significantly higher for patients with PAPP-A2 values above median than for patients with low PAPP-A2 levels (p=0.011). CONCLUSION: PAPP-A2 is increased in HD patients and interacts with PAPP-A on patients´ prognosis.

Comparison of DNA isolation using salting-out procedure and automated isolation (MagNA system).

Isolation of genomic DNA is a key step in genetic analysis. The aim of the study was to evaluate the suitability of isolation of DNA from peripheral blood with manual salting-out procedure and automated MagNA system under specific conditions. The impact of storage conditions, type of material (whole blood or blood cells), and method used for DNA extraction were evaluated in terms of DNA yield, its purity, and integrity. Fresh material, and material stored at 2-8°C for 1-4 weeks and frozen at -80°C were tested. For fresh samples, salting-out method gives higher yield than MagNA, irrespectively, on material used. Neither the yield of salting-out method nor its purity decreases during the storage of the samples in the fridge (2-8°C) during 4 weeks. Concerning MagNA, storage of blood cells in the fridge decreases the yield of DNA as well as its purity. For frozen samples, for whole blood, MagNA gives better results while for blood cells, salting-out method seems to be better. For fresh samples, salting-out method is the preferred one, and both whole blood and blood cells can be used. For frozen samples, the preferred method depends on the material.

Surface plasmon resonance biosensor for detection of pregnancy associated plasma protein A2 in clinical samples.

Pregnancy associated plasma protein A2 (PAPP-A2) is a metalloproteinase that plays multiple roles in fetal development and post-natal growth. Here we present a novel surface plasmon resonance (SPR) biosensor for the rapid and quantitative detection of PAPP-A2 in blood samples. This biosensor uses a single surface referencing approach and a sandwich assay with functionalized gold nanoparticles for signal enhancement. We demonstrate that this SPR biosensor enables the detection of PAPP-A2 in 30 % blood plasma at levels as low as 3.6 ng/mL. We also characterize the performance of the biosensor and evaluate its cross-reactivity to a PAPP-A analogue. Finally, we utilize this SPR biosensor for the detection of PAPP-A2 in blood serum from two groups of subjects: pregnant women and healthy non-pregnant women and men. Graphical Abstract Temporal sensor response corresponding to respective steps of the assay for detection of PAPP-A2 in buffer.

Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo.

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.

A Mechanism of O-Demethylation of Aristolochic Acid I by Cytochromes P450 and Their Contributions to This Reaction in Human and Rat Livers: Experimental and Theoretical Approaches.

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.

The influence of ochratoxin A on DNA adduct formation by the carcinogen aristolochic acid in rats.

Exposure to the plant nephrotoxin and carcinogen aristolochic acid (AA) leads to the development of AA nephropathy, Balkan endemic nephropathy (BEN) and upper urothelial carcinoma (UUC) in humans. Beside AA, exposure to ochratoxin A (OTA) was linked to BEN. Although OTA was rejected as a factor for BEN/UUC, there is still no information whether the development of AA-induced BEN/UUC is influenced by OTA exposure. Therefore, we studied the influence of OTA on the genotoxicity of AA (AA-DNA adduct formation) in vivo. AA-DNA adducts were formed in liver and kidney of rats treated with AA or AA combined with OTA, but no OTA-related DNA adducts were detectable in rats treated with OTA alone or OTA combined with AA. Compared to rats treated with AA alone, AA-DNA adduct levels were 5.4- and 1.6-fold higher in liver and kidney, respectively, of rats treated with AA combined with OTA. Although AA and OTA induced NAD(P)H: quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA-DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA-DNA adduct formation in liver and kidney of rats.

The effects of heavy metal ions, phthalates and ochratoxin A on oxidation of carcinogenic aristolochic acid I causing Balkan endemic nephropathy.

OBJECTIVES: Balkan endemic nephropathy (BEN) is a chronic progressive fibrosis associated with upper urothelial carcinoma (UUC). Aetiology of BEN is still not fully explained. Although carcinogenic aristolochic acid I (AAI) was proven as the major cause of BEN/UUC, this nephropathy is considered to be multifactorial. Hence, we investigated whether other factors considered as potential causes of BEN [a mycotoxin ochratoxin A (OTA), Cd, Pb, Se and As ions and organic compounds (i.e. phthalates) released from lignite deposits in BEN areas] can influence detoxication of AAI, whose concentrations are crucial for BEN development. METHODS: Oxidation of AAI to 8-hydroxyaristolochic acid I (AAIa) in the presence of Cd, Pb, Se, As ions, dibutylphthalate (DBP), butylbenzylphthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP) and OTA by rat liver microsomes was determined by HPLC. RESULTS: Only OTA, cadmium and selenium ions, and BBP inhibited AAI oxidation by rat liver microsomes. These compounds also inhibited activities of CYP1A1 and/or CYP2C6/11 catalysing AAI demethylation in rat livers. Therefore, these CYP inhibitions can be responsible for a decrease in AAIa formation. When the combined effects of these compounds were investigated, the most efficient inhibition was caused by OTA combined with BBP and selenium ions. CONCLUSION: The results show low effects of BBP, cadmium and selenium ions, and/or their combinations on AAI detoxication. No effects were produced by the other metal ions (Pb, As) and phthalates DBP and DEHP. This finding suggests that they do not influence AAI-mediated BEN development. In contrast, OTA might influence this process, by inhibition of AAI detoxication.

The effect of aristolochic acid I on expression of NAD(P)H:quinone oxidoreductase in mice and rats--a comparative study.

Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H: quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

The influence of dicoumarol on the bioactivation of the carcinogen aristolochic acid I in rats.

Aristolochic acid I (AAI) is the major toxic component of the plant extract AA, which leads to the development of nephropathy and urothelial cancer in human. Individual susceptibility to AAI-induced disease might reflect variability in enzymes that metabolise AAI. In vitro NAD(P)H: quinone oxidoreductase (NQO1) is the most potent enzyme that activates AAI by catalyzing formation of AAI-DNA adducts, which are found in kidneys of patients exposed to AAI. Inhibition of renal NQO1 activity by dicoumarol has been shown in mice. Here, we studied the influence of dicoumarol on metabolic activation of AAI in Wistar rats in vivo. In contrast to previous in vitro findings, dicoumarol did not inhibit AAI-DNA adduct formation in rats. Compared with rats treated with AAI alone, 11- and 5.4-fold higher AAI-DNA adduct levels were detected in liver and kidney, respectively, of rats pretreated with dicoumarol prior to exposure to AAI. Cytosols and microsomes isolated from liver and kidney of these rats were analysed for activity and protein levels of enzymes known to be involved in AAI metabolism. The combination of dicoumarol with AAI induced NQO1 protein level and activity in both organs. This was paralleled by an increase in AAI-DNA adduct levels found in ex vivo incubations with cytosols from rats pretreated with dicoumarol compared to cytosols from untreated rats. Microsomal ex vivo incubations showed a lower AAI detoxication to its oxidative metabolite, 8-hydroxyaristolochic acid (AAIa), although cytochrome P450 (CYP) 1A was practically unchanged. Because of these unexpected results, we examined CYP2C activity in microsomes and found that treatment of rats with dicoumarol alone and in combination with AAI inhibited CYP2C6/11 in liver. Therefore, these results indicate that CYP2C enzymes might contribute to AAI detoxication.

Dicoumarol inhibits rat NAD(P)H:quinone oxidoreductase in vitro and induces its expression in vivo.

OBJECTIVES: Dicoumarol is known to act as an inhibitor of NAD(P)H: quinone oxidoreductase (NQO1). This cytosolic reductase significantly contributes to the genotoxicity of the nephrotoxic and carcinogenic alkaloid aristolochic acid I (AAI). Aristolochic acid causes aristolochic acid nephropathy (AAN), and Balkan endemic nephropathy (BEN), as well as associated urothelial malignancies. NQO1 is the most efficient enzyme responsible for the reductive bioactivation of AAI to species forming covalent AAI-DNA adducts. However, it is still not known how dicoumarol influences the NQO1-mediated reductive bioactivation of AAI. METHODS: AAI-DNA adduct formation was determined by 32P-postlabeling. Expression of NQO1 mRNA and NQO1 protein was determined by real-time polymerase chain reaction and Western blotting, respectively. RESULTS: In this study, dicoumarol inhibited AAI bioactivation to form AAI-DNA adducts mediated by rat and human NQO1 in vitro as expected. We however, demonstrated that dicoumarol acts as an inducer of NQO1 in kidney and lung of rats treated with this NQO1 inhibitor in vivo, both at protein and activity levels. This NQO1 induction increased the potency of kidney cytosol to bioactivate AAI and elevated AAI-DNA adduct levels were found in ex-vivo incubations of AAI with renal cytosols and DNA. NQO1 mRNA levels were induced in liver only by dicoumarol. CONCLUSION: Our results indicate a dual role of dicoumarol in NQO1-mediated genotoxicty of AAI. It acts both as an NQO1 inhibitor mainly in vitro and as an NQO1 inducer if administered to rats.

NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I.

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)-the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1⁻/⁻, Cyp1a2⁻/⁻ and Cyp1a1/1a2⁻/⁻ knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Bioactivation versus detoxication of the urothelial carcinogen aristolochic acid I by human cytochrome P450 1A1 and 1A2.

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Role of cytochromes P450 in metabolism of carcinogenic aristolochic acid I: evidence of their contribution to aristolochic acid I detoxication and activation in rat liver.

OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.

Role of P450 1A1 and P450 1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: studies in Cyp1a1-/-, Cyp1a2-/-, and Cyp1a1/1a2-/- mice.

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: studies with the hepatic NADPH:cytochrome P450 reductase null (HRN) mouse model.

Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.