Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells

Schiwy, Andreas; Brinkmann, Markus; Thiem, Ines; Guder, Gabriele; Winkens, Kerstin; Eichbaum, Kathrin; Nüßer, Leonie; Thalmann, Beat; Buchinger, Sebastian; Reifferscheid, Georg; Seiler, Thomas‐Benjamin; Thoms, Brigitte; Hollert, Henner

doi:10.1038/nprot.2015.108

Cited by 42 publications

(32 citation statements)

References 53 publications

(49 reference statements)

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“…Metabolic activation could therefore, in principle, occur in all of the reported ponatinib target tissues. CYP1A1 levels are constitutively very low, but are highly inducible on activation of the aryl hydrocarbon receptor by compounds including polycyclic aromatic hydrocarbons found in cigarette smoke ( Schiwy et al, 2015 ). It is interesting to note that hypertension or vaso-occlusive disease observed following ponatinib administration has been associated with smoking ( Jain et al, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites

Lin

Kostov

Huang

et al. 2017

J Pharmacol Exp Ther

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Ponatinib, a pan-BCR-ABL tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia (CML), causes severe side effects including vascular occlusions, pancreatitis, and liver toxicity, although the underlying mechanisms remain unclear. Modifications of critical proteins through reactive metabolites are thought to be responsible for a number of adverse drug reactions. In vitro metabolite screening of ponatinib with human liver microsomes and glutathione revealed unambiguous signals of ponatinib-glutathione (P-GSH) adducts. Further profiling of human cytochrome P450 (P450) indicated that CYP1A1 was the predominant P450 enzyme driving this reaction. P-GSH conjugate formation paralleled the disappearance of hydroxylated ponatinib metabolites, suggesting the initial reaction was epoxide generation. Mouse glutathione S-transferase p1 (mGstp1) further enhanced P-GSH adduct formation in vitro. Ponatinib pharmacokinetics were determined in vivo in wild-type (WT) mice and mice humanized for CYP1A1/2 and treated with the CYP1A1 inducers 2,3,7,8-tetrachlorodibenzodioxin or 3-methylcholanthrene. Ponatinib exposure was significantly decreased in treated mice compared with controls (7.7- and 2.2-fold for WT and humanized CYP1A1/2, respectively). Interestingly, the P-GSH conjugate was only found in the feces of CYP1A1-induced mice, but not in control animals. Protein adducts were also identified by liquid chromatography–tandem mass spectrometry analysis of mGstp1 tryptic digests. These results indicate that not only could CYP1A1 be involved in ponatinib disposition, which has not been previously reported, but also that electrophilic intermediates resulting from CYP1A1 metabolism in normal tissues may contribute to ponatinib toxicity. These data are consistent with a recent report that CML patients who smoke are at greater risk of disease progression and premature death.

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Section: Discussionmentioning

confidence: 99%

Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites

Lin

Kostov

Huang

et al. 2017

J Pharmacol Exp Ther

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“…18 Briefly, a non-fluorescent CYP1A1 substrate, 7-ethoxyresorufin, is applied on cultured hepatocytes, and then, the fluorescence of the product, resorufin, is analysed using a plate reader. 19…”

Section: Enzymatic Activity Of Cyp1a1mentioning

confidence: 99%

Microcomedones in non‐lesional acne prone skin New orientations on comedogenesis and its prevention

Fontao

Engelbrechten

Seilaz

et al. 2019

Acad Dermatol Venereol

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Background In non‐lesional skin of acne patients, cyanoacrylate skin surface stripping can harvest a structure called microcomedone (MC) which is the earliest phase of comedogenesis; the root of any subsequent clinical lesion and a target for the prevention of acne relapses. More information is needed on the putative biochemical contributors (biomarkers) of comedogenesis expressed in MC. Methods Proteins expressed in MC were screened by proteomics, immunohistochemistry and Western blotting. The in vitro effects of a comedolytic Silybum marianum fruit extract (SMFE) were studied in sebocyte cultures by RNA‐Seq and modulation of CYP1A1 by qPCR and enzymatic activity. MC severity was correlated to lesions counts and keratin expression during 48 weeks in 23 acne patients using a topical comedolytic formulation containing SMFE. Results Two infundibular keratins, K75 and K79, co‐localized in MC with the sebocyte progenitor cell marker LRIG1 and were used as a biomarker of comedogenesis for the follow‐up of patients. In cultured sebocytes exposed to SMFE (i) transcriptomic analysis showed an up‐regulation by a factor of 15 of RNA coding for K75 and (ii) the gene expression and catalytic activity of CYP1A1 under exposure to dioxin was decreased. In the acne patients using SMFE, the MC index in non‐lesional skin decreased over time and remained until the 48th week, significantly lower than that of the first week. There was a high correlation between the decrease of MC index and the decrease and stability of the clinical lesions counts over time. Importantly, a low MC index status was found to be associated with a significant higher K75 expression in microcomedones. Discussion These observations provide new orientations on the mechanism of comedogenesis and its prevention. Maintaining a low MC status in non‐lesional skin is a sound target for the prevention of acne relapse and a good sentinel of acne remissions.

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“…The rCYP1A1 activity of the monolith-based IMERs (Fig. 1 d) was determined with an ethoxyresorufin-O-deethylase (EROD) assay [ 27 – 29 ]. The EROD assay was performed with 2 μM ethoxyresorufin as the substrate and 0.5 mM NADPH as the co-substrate, both dissolved in 0.1 M potassium phosphate buffer (pH 7.4) containing 5 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research

et al. 2018

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We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1280-7) contains supplementary material, which is available to authorized users.

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Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells

Cited by 42 publications

References 53 publications

Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites

Novel Pathways of Ponatinib Disposition Catalyzed By CYP1A1 Involving Generation of Potentially Toxic Metabolites

Microcomedones in non‐lesional acne prone skin New orientations on comedogenesis and its prevention

Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research

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