Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with...
Transcript of Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with...
![Page 1: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/1.jpg)
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare
Substanzen
Hans F Merk
RWTH Aachen University
hansmerkpostrwth-aachende
1968 Beginn der KoKo
bull Praumlsident USA Lyndon B Johnson
bull Bundespraumlsident Heinrich Luumlbke ndash Gustav Heinemann
bull Bundeskanzler Kurt Georg Kiesinger
bull Nobelpreis 1977 Robert W Holley Isolierung einer t-RNA Marshall W Nirenberg PolyU-Experiment Har G Khorana gelang 1970 als erstem die kuumlnstliche Synthese eines Gens
Asthma (IgE) durch kleinmolekulare Substanzen nur nach Sensibilierung (mit Ekzemreaktion) uumlber die Haut
J Pauluhn Toxicology 319 (2014) 10 -22
Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria
recipient of the award in 2003)
Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical
backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959
Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8
1966 Human repeated insult patch test Albert Kligman
J invest Dermatol 47 (1966) 393 - 409
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 2: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/2.jpg)
1968 Beginn der KoKo
bull Praumlsident USA Lyndon B Johnson
bull Bundespraumlsident Heinrich Luumlbke ndash Gustav Heinemann
bull Bundeskanzler Kurt Georg Kiesinger
bull Nobelpreis 1977 Robert W Holley Isolierung einer t-RNA Marshall W Nirenberg PolyU-Experiment Har G Khorana gelang 1970 als erstem die kuumlnstliche Synthese eines Gens
Asthma (IgE) durch kleinmolekulare Substanzen nur nach Sensibilierung (mit Ekzemreaktion) uumlber die Haut
J Pauluhn Toxicology 319 (2014) 10 -22
Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria
recipient of the award in 2003)
Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical
backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959
Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8
1966 Human repeated insult patch test Albert Kligman
J invest Dermatol 47 (1966) 393 - 409
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 3: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/3.jpg)
Asthma (IgE) durch kleinmolekulare Substanzen nur nach Sensibilierung (mit Ekzemreaktion) uumlber die Haut
J Pauluhn Toxicology 319 (2014) 10 -22
Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria
recipient of the award in 2003)
Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical
backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959
Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8
1966 Human repeated insult patch test Albert Kligman
J invest Dermatol 47 (1966) 393 - 409
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 4: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/4.jpg)
Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria
recipient of the award in 2003)
Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical
backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959
Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8
1966 Human repeated insult patch test Albert Kligman
J invest Dermatol 47 (1966) 393 - 409
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 5: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/5.jpg)
1966 Human repeated insult patch test Albert Kligman
J invest Dermatol 47 (1966) 393 - 409
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 6: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/6.jpg)
1966 Human repeated insult patch test Albert Kligman
1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 7: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/7.jpg)
1968 Beginn der KoKo
1968 1969
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 8: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/8.jpg)
First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 9: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/9.jpg)
The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin
DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 10: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/10.jpg)
RWTH Aachen1995
Cytochrom P450 Haut
JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 11: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/11.jpg)
The skin as a metabolizing organ of
pro-haptens
Cytochrom P450
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 12: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/12.jpg)
Activation of prohaptens is mediated by CYPs expressed in skin cells
CYP amount
(pmol)
Skin specific
rhCYP cocktail
CYP1A1 36
CYP1B1 20
CYP2B6 0035
CYP2E1 11
CYP3A5 56
total CYP amount 22
Stratum corneum epidermis
PROHAPTEN
Exte
rn
a
HAPTEN ANTIGEN
PROTEIN ENZYMATIC ACTIVATION
CYP
CYP
content
(pmol)
Liver-like
rhCYP cocktail
CYP1A2 20
CYP2C9 17
CYP2C19 80
CYP2D6 30
CYP2E1 11
CYP3A4 45
Total CYP content 104
(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12
12
101
2 ss
nn
X
X
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 13: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/13.jpg)
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 14: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/14.jpg)
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 15: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/15.jpg)
Autooxidation and CYP-metabolism important in sensitization to geraniol
Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27
Neral
Geranial
67-Epoxygeraniol
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 16: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/16.jpg)
Skin sensitization and Regulation
- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 17: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/17.jpg)
The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers
ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2
A Natsch Toxicological Sciences Volume 113 2010 284ndash292
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 18: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/18.jpg)
KeratinoSensreg-Assay
Luciferase Gene SV40 ARE
Keap1
Nrf2
Hapten
Luminescence Luciferase Gene SV40 ARE
Keap1
Nrf2
Nrf2
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 19: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/19.jpg)
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
2 days
30min
KeratinoSens-Cells
Viability
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 20: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/20.jpg)
Modified KeratinoSensreg Assay
Hapten Pro-
hapten
Luminescence
Pro-hapten
S9
CYP Cocktail CYP1A1
CYP1B1
CYP2B6
CYP2E1
CYP3A5
2 days
30min
KeratinoSens-Cells
Viability
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 21: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/21.jpg)
KeratinoSens with Carvoxime
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 22: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/22.jpg)
Eugenol - Creosol
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 23: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/23.jpg)
KeratinoSens mit Creosol
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 24: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/24.jpg)
Methods 2
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 25: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/25.jpg)
HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)
Creo
Creo
Creo
12 benzoquinone-hapten-protein-adduct
hapten-protein-adduct
Methandiol (monohydrated formaldehyd)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 26: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/26.jpg)
Bioactivationreactivity of creosol-13C2 in 3D skin equivalents
Epidermis + Dermis
Dermis
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 27: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/27.jpg)
Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen
Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1
S-4-Methoxydalbergion 1
Obtusachinon 1
S-44-Dimethoxydalbergion 1
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion
![Page 28: Haut: Zielorgan allergischer Reaktionen auf ...€¦ · Steigleder GK Personal experiences with Stephen Rothman, M.D. J Am Acad Dermatol. 1988 Sep;19(3):596-8. ... Volume 113, 2010,](https://reader034.fdocument.pub/reader034/viewer/2022051813/60342d099e7d2941586ae4c1/html5/thumbnails/28.jpg)
Haut Zielorgan allergischer Reaktionen auf
kleinmolekulare Substanzen
0
5
10
15
20
25
(S)-44DMD [R]-4-MD (S)-4-MD
10(-8)M
5x10(-8)M
10(-7)M
5x10(-7)M
10(-6)M
5x10(-6)
10(-5)M
SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation
SI
Klinik fuumlr Dermatologie amp Allergologie
PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108
M
DMD Dimethoxydalbergion MD Methoxydalbergion