1. A Skin-Like Cytochrome P450 Cocktail Activates Prohaptens to Contact Allergenic Metabolites 
Moa Andresen Bergström, Hagen Ott, Anna Carlsson, Mark Neis, Gabriele Zwadlo-Klarwasser, Charlotte A.M. Jonsson, Hans F. Merk, Ann-Therese Karlberg, Jens M. Baron 
Journal of Investigative Dermatology 
Volume 127, Issue 5, Pages (May 2007)
DOI: /sj.jid
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
CYP-mediated metabolism of the diene to the endocyclic and exocyclic epoxide followed by hydrolysis of the exocyclic epoxide to a diol.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Metabolic activation of the diene in liver microsomal incubations. Detected amounts of the endocyclic epoxide (▪) and the diol of the exocyclic epoxide (□) in incubations with the (a) mouse and (b) human liver microsomes. The microsomal incubations were performed using human or mouse liver microsomes (0.5mg of protein), substrate (200μM), potassium phosphate buffer (100mM, pH 7.4), and a NADPH-regenerating system in a total volume of 500μl. The incubations were terminated after 15, 30, or 60minutes and analyzed using GC/MS and LC/MS. The detections limits for the endocyclic epoxide and the diol of the exocyclic epoxide are 5 and 25pmol (total amount in incubation mixture), respectively. The figure shows the total amounts formed of the metabolites in the incubation mixtures, presented as means of two individual incubations. ND=not detected.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Metabolic activation of the diene in rhCYP cocktail incubations. Detected amounts of the endocyclic epoxide (▪) and the diol of the exocyclic epoxide (□) in incubations with the (a) liver and (b) skin system. The incubations were performed using the liver-like and skin-like cocktail (prepared by mixing recombinantly expressed CYP enzymes according to Table 1), substrate (200μM), HEPES buffer (50mM, pH 7.4), MgCl2 (30mM), and NADPH (1mM) in a total volume of 500μl. The incubations were terminated after 15, 30, or 60minutes and analyzed using GC/MS and LC/MS. The detections limits for the endocyclic epoxide and the diol of the exocyclic epoxide are 5 and 25pmol (total amount in incubation mixture), respectively. The figure shows the total amounts formed of the metabolites in the incubation mixtures, presented as means of two individual incubations. TD=traces detected.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Metabolic activation of the diene in incubations with rhCYP enzymes. Detected amounts of the endocyclic epoxide (▪) and the diol of the exocyclic epoxide (□) in incubations with single recombinant CYP enzymes present in the (a) liver- and (b) skin-like rhCYP cocktails. The incubations were performed using recombinant human CYPs (20pmol; CYP1A1, 3A5, 1B1, 2B6, 2E1, 1A2, 3A4, 2C9, 2C19, or 2D6), substrate (200mM), HEPES buffer (50mM, pH 7.4), MgCl2 (30mM), and NADPH (1mM) in a total volume of 500μl. The incubations were terminated after 30minutes and analyzed using GC/MS and LC/MS. The detections limits for the endocyclic epoxide and the diol of the exocyclic epoxide are 5 and 25pmol (total amount in incubation mixture), respectively. The figure shows the total amounts formed of the metabolites in the incubation mixtures, presented as means of two individual incubations. ND=not detected.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
In vitro sensitizing capacity of the diene, the epoxides and the diol in a DC assay. Immature human DCs were incubated with 1: diene (100μM), 2: exocyclic epoxide (200μM), 3: endocyclic epoxide (200μM), and 4: major diol of the exocyclic epoxide (200μM). 5: 2,4,6-Trinitrobenzene sulfonic acid (200μg/ml) was used as a positive control and 6: DMSO (0.1%) as well as 7: SDS (5μg/ml) as negative controls. The cells were harvested after incubation for 30hours and the mRNA was isolated and analyzed by quantitative reverse transcriptase-PCR. The measured stimulation of IL-8 was normalized relative to medium-treated controls. The data shown are medians of four experiments and the results are expressed as x-fold upregulation compared to medium-treated controls.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions