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Phototoxicity in vitro: Investigation of photoreactions in the skin using the reconstructed epidermis “Epidermal Skin Test 1000” (EST-1000)
Abstracts / Toxicology Letters 180S (2008) S32–S246
antioxidants and beneficial on breast cancer, prostate cancer, menopausal symptoms, osteoporosis and cardiovascular disease. On the other hand several studies have demonstrated that phytoestrogens exert biphasic effects; depending on dose they can also act as prooxidants and procarcinogens. In the present study the conditions which the biphasic effects of quercetin and genistein as antioxidant and prooxidant occur have been investigated in vitro. To achieve this goal, human red blood cells (RBC) and rat liver microsomes are used as biological models and incubated in vitro with various concentrations of quercetin or genistein in the presence or absence of transition metals, azide and hydrogen peroxide. Prooxidant and/or antioxidant effects of the compounds are evaluated by determination of sulfhydryl levels as an indicator of antioxidant defense and malondialdehyde levels as an endpoint of membrane lipid peroxidation. Beside investigating effects of ROS, a fluorescent probe, 2 -,7 -dichlorodihydrofluorescein diacetate, was used to indicate the formation of reactive species by quercetin and genistein. Various concentrations of genistein and quercetin were able to prevent the decrease of sulfhydryl levels in the presence of transition metals alone and in conjunction with azide. However this antioxidant effect was found to be independent from genistein concentration in the present RBC model. Antioxidant effects of quercetin and genistein rather than a prooxidant effect was observed in the present RBC model and incubation conditions. Results from the experiments performed in microsomes will be further discussed. doi:10.1016/j.toxlet.2008.06.424 V34 Drug metabolizing enzyme activity in human in vitro dermal (EpiDerm) and airway (EpiAirway) epithelial models Patrick Hayden ∗ , Jennifer Bolmarcich, George Stolper, Helena Kandarova, Mitchell Klausner
Jackson, Gina
MatTek Corporatin, Ashland, MA, United States Human dermal and airway epithelia contain xenobiotic metabolizing enzymes (XME) that can cause biotransformation of drugs and environmental/occupational chemicals, resulting in altered drug activity or formation of toxic/mutagenic metabolites. The present work evaluated expression of XMEs in highly differentiated in vitro models of human dermal (EpiDerm) and airway (EpiAirway) epithelia. RT-PCR experiments were conducted to evaluate baseline and inducible expression of cytochrome P450 (CYP) isoforms in the epithelial cultures. EpiAirway cultures constitutively expressed CYP1A1 (weak), CYP1B1, CYP2A6, CYP2B6 (weak), CYP2C8 (weak), CYP2C19, CYP2D6, CYP2E1 and CYP3A5, while CYP3A4 and 3A7 were not detected. 3-Methylcholanthrene (3MC) strongly increased expression of CYP1A1 and slightly increased CYP2B6 and CYP2C8 expression in EpiAirway. In EpiDerm, CYP1B1, CYP2C19, CYP2D6, CYP3A4 (weak) and CYP3A5 were constitutively expressed. 3-Methylcholanthrene (3MC) strongly increased expression of CYP1A1 and CYP1B1 in EpiDerm. Enhanced metabolism of the CYP1A1 and CYP1B1 substrate ethoxyresorufin confirmed increased activity following treatment with 3MC. Thus CYP expression in EpiAirway and EpiDerm showed a high concordance with CYP expression reported for in vivo human airway and dermal epithelia. Total Glutathione S-transferase (GST) activity in the epithelial models was also evaluated by measuring conjugation of glutathione with 1-chloro2,4-dinitrobenzene and UDP-glucuronyltransferase activity was determined by 4-methylumbellipherone conjugation. High baseline GST and UDP-glucuronyltransferase activity in both models was not further enhanced by 3MC treatment. The results demon-
S105
strate that the EpiDerm and EpiAirway in vitro human epithelial models possess in vivo-like XME activities and may thus be useful for evaluating epithelial metabolism of drugs and environmental/occupational chemicals. doi:10.1016/j.toxlet.2008.06.425 V35 Healing of dermal burn wounds in the EpiDerm-FTTM in vitro human skin model Patrick Hayden ∗ , Gina Stolper, Carolyn Cooney, Helena Kandarova, Mitchell Klausner MatTek Corporation, Ashland, MA, United States Dermal wound healing involves interactions between dermal fibroblasts and epidermal keratinocytes, as well as cell–extracellular matrix interactions. The current poster describes wound-healing experiments conducted with a full-thickness in vitro human skin model (EpiDerm-FTTM ). Normal human epidermal keratinocytes (KC) and dermal fibroblasts (FB) were cultured to produce the highly differentiated full-thickness skin model. Histological examination of EpiDerm-FTTM shows a collagen dermis populated by viable FB and an epidermis of stratified KC including basal, spinous, granular and stratum corneum components. Small burn wounds of several millimeters in diameter were induced in the epithelial model by means of a battery-operated cauterizer. The wounded EpiDerm-FTTM cultures were fixed at various time points and H&E stained paraffin sections were prepared to evaluate the wound and the wound healing process. Immediately after burn wounding, necrotic epithelium and denatured collagen dermal matrix were evident. Within one day, the denatured collagen matrix began to degrade and epithelial KC were observed migrating inward from the wound edges. Over a time course of seven days, migrating KC repopulated the wounded area to form a fully covered epithelium. Dermal fibroblasts were also observed to be proliferating within the wound area and generating new dermal matrix material. These results demonstrate that the EpiDermFTTM model may be useful for applications designed to elucidate dermal–epidermal interactions during wound healing and to evaluate the role of specific growth factors or new therapeutics in the dermal wound healing process. doi:10.1016/j.toxlet.2008.06.426 V36 Phototoxicity in vitro: Investigation of photoreactions in the skin using the reconstructed epidermis “Epidermal Skin Test 1000” (EST-1000) Jens Hoffmann 1,∗ , Eckhard Heisler 2 , Sabine Weimans 2 , Astrid Thiemann 2 , Andreas Schnurstein 2 , Horst W. Fuchs 1 1
CellSystems Biotechnologie VErtrieb GmbH, St. Katharinen, Germany, 2 EVONIK Stockhausen GmbH, Krefled, Germany In 1998, the 3T3 Neutral Red Uptake Photocytotoxicity Assay according to Spielmann et al. has been established as a suitable in vitro test method for the evaluation of risks derived from photoreactive compounds (OECD Testguideline 432). However this test method works best with soluble and non-volatile. In different studies on photocytotoxicity it has recently been demonstrated that reconstructed human skin is a potent tool for in vitro phototoxicity testing. In comparison to the murine 3T3 cell line these models provide several advantages. First of all human skin models are capable to reflect topical exposure conditions followed by UV irradiation.
S106
Abstracts / Toxicology Letters 180S (2008) S32–S246
Furthermore, test methods based on tissue models are not limited to a rather small subset of chemicals since solid and insoluble test compounds can be applied very easily to the surface of an epidermal model. Additionally epidermal models like Epidermal Skin Test 1000 (EST-1000, CellSystems, Germany) are derived from Keratinocytes of human origin and therefore inter species variation can be excluded. In this study EST-1000 has been topically exposed to photocytotoxic compounds like Chlorpromazine followed by defined UV irradiation. Depending on their concentration the photocytotoxic potential of these compounds has been monitored. Additionally we demonstrated that the application of skin care products like sun blockers reduced the toxic effects of the test chemicals. From our results we conclude that EST-1000 is a suitable in vitro tool for the evaluation of the phototoxic potential of chemicals as well as for the efficacy testing of products from cosmetic skin care products. doi:10.1016/j.toxlet.2008.06.427 V37 Effects of mitochondrial toxic drugs on mitochondrial DNA gene expression in HepG2 cells Doris Höschele ∗ , Martina Wiertz, Inmaculada Garcia Moreno Federal Institute for Drugs and Medical Devices, 53175 Bonn, Germany Mitochondrial toxicity is caused by different classes of drugs including the antiretroviral nucleoside reverse transcriptase inhibitors zalcitabine (ddC) and zidovudine (AZT), the DNA intercalating agent ethidium bromide (EtBr), the antibacterial drugs nalidixin acid (NAL) and chloramphenicol (CAP). Although several studies investigated their effects on mitochondrial function and/or mitochondrial DNA (mtDNA) production, little is known about the influence on mtDNA gene expression. This study aims to investigate effects of these drugs on gene expression of all 13 mtDNA-encoded proteins in HepG2 cells in parallel to mtDNA content and cellular lactate production. HepG2 cells were treated over 14 days with 1, 10, and 100 M ddC, AZT, NAL, or CAP and 50 and 100 ng/mL EtBr. MtDNA gene expression and mtDNA content were analysed by real-time PCR. Mitochondrial function was determined by measuring cellular lactate production. All drugs altered mtDNA gene expression. DdC induced increase in gene expression at 1 M for most genes but a decrease at 100 M. Gene expression at 10 M was variable for individual genes. AZT treatment reduced gene expression at 1 and 10 M. At 100 M weaker or no reduction was found. For EtBr, NAL and CAP gene expression decreased concentration-dependently for the majority of genes. Alterations in gene expression were accompanied by increases in cellular lactate production and changes in mtDNA content. In conclusion, mtDNA gene expression in HepG2 cells alters in response to treatment with mitochondrial toxic drugs. Further investigation is needed to find out whether these effects are direct or indirect effects. doi:10.1016/j.toxlet.2008.06.428
V38 ECVAM activities in the validation of alternatives for endocrine disruptor testing Miriam Jacobs ∗ , Susanne Bremer, Thomas Hartung, Patricia Pazos, Cristian Pellizzer ECVAM, IHCP EC JRC, Ispra, Italy Legislation or draft proposals in for instance Europe, Japan and the USA require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances and may be putative endocrine disruptors (EDs). To address these requirements and the need to develop alternatives to in vivo testing as part of a tiered testing strategy, ECVAM is involved in European and international collaborations including the OECD test guideline programme. As part of the EU ReProTect Integrated Project, and under the auspices of the OECD, ECVAM is involved in the management of the (pre)validation of several Estrogen (ER) and Androgen receptor (AR) transactivation and ligand binding in vitro assays. Following the ECVAM modular approach to validation and the OECD Guidance Document 34, these tests are have been assessed for modules 1–4, test definition, within laboratory definition, transferability, and between laboratory variability. Most are now are entering module 5 to assess predictive capacity. In addition ECVAM is collaborating with ICCVAM and JaCVAM in the validation of the LUMI cell ER transactivation assays, and with JaCVAM for the validation of the antagonist HeLa ER in vitro assay. doi:10.1016/j.toxlet.2008.06.429 V39 Modeling inflammation–drug interactions lymphocytes–hepatocyte co-culture system
in
vitro:
A
Yong Pil Hwang, Eun Hee Han, Hye Gwang Jeong ∗ Department of Pharmacy, Chosun University, Gwangju, Republic of Korea Xenobiotic–inflammation interactions lead to hepatotoxicity in vivo. The liver immune system comprises a major component of the innate immune system. Lymphocytes are the major cells of the innate immune response; by producing cytokines/chemokines and cytotoxic lysis of transformed cells. Selected xenobiotic agents (acetaminophen, APAP; concanavalin A, Con A; fumonisin B1, FB1) for which this occurs were evaluated for ability to elicit the release of lymphocytes-derived inflammatory mediators. In the current study, cellular interactions between lymphocytes (LCs) and hepatocytes (HPCs) in xenobiotics-induced hepatotoxicity were investigated, using co-cultures of mouse splenic lymphocytes and human hepatoma HepG2 cells. Treatment of the co-cultures with xenobiotics produced hepatotoxicity, whereas either LCs or HPCs cultures alone showed no response to xenobiotics. Expression of TNF-alpha and interferon-gamma was increased in co-cultures but not in the individual cell cultures. These results indicated that LCs and HPCs interact in response to xenobiotics and xenobiotics potentiates the proapoptotic cytokines production in LCs, which may have implications in making HPCs responsive to cytotoxicity of xenobiotics in the liver. In summary, results from these studies using co-cultures of LCs and HPCs qualitatively mirrored data from whole animal studies. Accordingly, this model might have particular utility for assessing drug-inflammation interactions as they relate to inflammatory mediator production. Moreover, this system is amenable to exploration of mechanisms of xenobiotic–inflammation interaction.
antioxidants and beneficial on breast cancer, prostate cancer, menopausal symptoms, osteoporosis and cardiovascular disease. On the other hand several studies have demonstrated that phytoestrogens exert biphasic effects; depending on dose they can also act as prooxidants and procarcinogens. In the present study the conditions which the biphasic effects of quercetin and genistein as antioxidant and prooxidant occur have been investigated in vitro. To achieve this goal, human red blood cells (RBC) and rat liver microsomes are used as biological models and incubated in vitro with various concentrations of quercetin or genistein in the presence or absence of transition metals, azide and hydrogen peroxide. Prooxidant and/or antioxidant effects of the compounds are evaluated by determination of sulfhydryl levels as an indicator of antioxidant defense and malondialdehyde levels as an endpoint of membrane lipid peroxidation. Beside investigating effects of ROS, a fluorescent probe, 2 -,7 -dichlorodihydrofluorescein diacetate, was used to indicate the formation of reactive species by quercetin and genistein. Various concentrations of genistein and quercetin were able to prevent the decrease of sulfhydryl levels in the presence of transition metals alone and in conjunction with azide. However this antioxidant effect was found to be independent from genistein concentration in the present RBC model. Antioxidant effects of quercetin and genistein rather than a prooxidant effect was observed in the present RBC model and incubation conditions. Results from the experiments performed in microsomes will be further discussed. doi:10.1016/j.toxlet.2008.06.424 V34 Drug metabolizing enzyme activity in human in vitro dermal (EpiDerm) and airway (EpiAirway) epithelial models Patrick Hayden ∗ , Jennifer Bolmarcich, George Stolper, Helena Kandarova, Mitchell Klausner
Jackson, Gina
MatTek Corporatin, Ashland, MA, United States Human dermal and airway epithelia contain xenobiotic metabolizing enzymes (XME) that can cause biotransformation of drugs and environmental/occupational chemicals, resulting in altered drug activity or formation of toxic/mutagenic metabolites. The present work evaluated expression of XMEs in highly differentiated in vitro models of human dermal (EpiDerm) and airway (EpiAirway) epithelia. RT-PCR experiments were conducted to evaluate baseline and inducible expression of cytochrome P450 (CYP) isoforms in the epithelial cultures. EpiAirway cultures constitutively expressed CYP1A1 (weak), CYP1B1, CYP2A6, CYP2B6 (weak), CYP2C8 (weak), CYP2C19, CYP2D6, CYP2E1 and CYP3A5, while CYP3A4 and 3A7 were not detected. 3-Methylcholanthrene (3MC) strongly increased expression of CYP1A1 and slightly increased CYP2B6 and CYP2C8 expression in EpiAirway. In EpiDerm, CYP1B1, CYP2C19, CYP2D6, CYP3A4 (weak) and CYP3A5 were constitutively expressed. 3-Methylcholanthrene (3MC) strongly increased expression of CYP1A1 and CYP1B1 in EpiDerm. Enhanced metabolism of the CYP1A1 and CYP1B1 substrate ethoxyresorufin confirmed increased activity following treatment with 3MC. Thus CYP expression in EpiAirway and EpiDerm showed a high concordance with CYP expression reported for in vivo human airway and dermal epithelia. Total Glutathione S-transferase (GST) activity in the epithelial models was also evaluated by measuring conjugation of glutathione with 1-chloro2,4-dinitrobenzene and UDP-glucuronyltransferase activity was determined by 4-methylumbellipherone conjugation. High baseline GST and UDP-glucuronyltransferase activity in both models was not further enhanced by 3MC treatment. The results demon-
S105
strate that the EpiDerm and EpiAirway in vitro human epithelial models possess in vivo-like XME activities and may thus be useful for evaluating epithelial metabolism of drugs and environmental/occupational chemicals. doi:10.1016/j.toxlet.2008.06.425 V35 Healing of dermal burn wounds in the EpiDerm-FTTM in vitro human skin model Patrick Hayden ∗ , Gina Stolper, Carolyn Cooney, Helena Kandarova, Mitchell Klausner MatTek Corporation, Ashland, MA, United States Dermal wound healing involves interactions between dermal fibroblasts and epidermal keratinocytes, as well as cell–extracellular matrix interactions. The current poster describes wound-healing experiments conducted with a full-thickness in vitro human skin model (EpiDerm-FTTM ). Normal human epidermal keratinocytes (KC) and dermal fibroblasts (FB) were cultured to produce the highly differentiated full-thickness skin model. Histological examination of EpiDerm-FTTM shows a collagen dermis populated by viable FB and an epidermis of stratified KC including basal, spinous, granular and stratum corneum components. Small burn wounds of several millimeters in diameter were induced in the epithelial model by means of a battery-operated cauterizer. The wounded EpiDerm-FTTM cultures were fixed at various time points and H&E stained paraffin sections were prepared to evaluate the wound and the wound healing process. Immediately after burn wounding, necrotic epithelium and denatured collagen dermal matrix were evident. Within one day, the denatured collagen matrix began to degrade and epithelial KC were observed migrating inward from the wound edges. Over a time course of seven days, migrating KC repopulated the wounded area to form a fully covered epithelium. Dermal fibroblasts were also observed to be proliferating within the wound area and generating new dermal matrix material. These results demonstrate that the EpiDermFTTM model may be useful for applications designed to elucidate dermal–epidermal interactions during wound healing and to evaluate the role of specific growth factors or new therapeutics in the dermal wound healing process. doi:10.1016/j.toxlet.2008.06.426 V36 Phototoxicity in vitro: Investigation of photoreactions in the skin using the reconstructed epidermis “Epidermal Skin Test 1000” (EST-1000) Jens Hoffmann 1,∗ , Eckhard Heisler 2 , Sabine Weimans 2 , Astrid Thiemann 2 , Andreas Schnurstein 2 , Horst W. Fuchs 1 1
CellSystems Biotechnologie VErtrieb GmbH, St. Katharinen, Germany, 2 EVONIK Stockhausen GmbH, Krefled, Germany In 1998, the 3T3 Neutral Red Uptake Photocytotoxicity Assay according to Spielmann et al. has been established as a suitable in vitro test method for the evaluation of risks derived from photoreactive compounds (OECD Testguideline 432). However this test method works best with soluble and non-volatile. In different studies on photocytotoxicity it has recently been demonstrated that reconstructed human skin is a potent tool for in vitro phototoxicity testing. In comparison to the murine 3T3 cell line these models provide several advantages. First of all human skin models are capable to reflect topical exposure conditions followed by UV irradiation.
S106
Abstracts / Toxicology Letters 180S (2008) S32–S246
Furthermore, test methods based on tissue models are not limited to a rather small subset of chemicals since solid and insoluble test compounds can be applied very easily to the surface of an epidermal model. Additionally epidermal models like Epidermal Skin Test 1000 (EST-1000, CellSystems, Germany) are derived from Keratinocytes of human origin and therefore inter species variation can be excluded. In this study EST-1000 has been topically exposed to photocytotoxic compounds like Chlorpromazine followed by defined UV irradiation. Depending on their concentration the photocytotoxic potential of these compounds has been monitored. Additionally we demonstrated that the application of skin care products like sun blockers reduced the toxic effects of the test chemicals. From our results we conclude that EST-1000 is a suitable in vitro tool for the evaluation of the phototoxic potential of chemicals as well as for the efficacy testing of products from cosmetic skin care products. doi:10.1016/j.toxlet.2008.06.427 V37 Effects of mitochondrial toxic drugs on mitochondrial DNA gene expression in HepG2 cells Doris Höschele ∗ , Martina Wiertz, Inmaculada Garcia Moreno Federal Institute for Drugs and Medical Devices, 53175 Bonn, Germany Mitochondrial toxicity is caused by different classes of drugs including the antiretroviral nucleoside reverse transcriptase inhibitors zalcitabine (ddC) and zidovudine (AZT), the DNA intercalating agent ethidium bromide (EtBr), the antibacterial drugs nalidixin acid (NAL) and chloramphenicol (CAP). Although several studies investigated their effects on mitochondrial function and/or mitochondrial DNA (mtDNA) production, little is known about the influence on mtDNA gene expression. This study aims to investigate effects of these drugs on gene expression of all 13 mtDNA-encoded proteins in HepG2 cells in parallel to mtDNA content and cellular lactate production. HepG2 cells were treated over 14 days with 1, 10, and 100 M ddC, AZT, NAL, or CAP and 50 and 100 ng/mL EtBr. MtDNA gene expression and mtDNA content were analysed by real-time PCR. Mitochondrial function was determined by measuring cellular lactate production. All drugs altered mtDNA gene expression. DdC induced increase in gene expression at 1 M for most genes but a decrease at 100 M. Gene expression at 10 M was variable for individual genes. AZT treatment reduced gene expression at 1 and 10 M. At 100 M weaker or no reduction was found. For EtBr, NAL and CAP gene expression decreased concentration-dependently for the majority of genes. Alterations in gene expression were accompanied by increases in cellular lactate production and changes in mtDNA content. In conclusion, mtDNA gene expression in HepG2 cells alters in response to treatment with mitochondrial toxic drugs. Further investigation is needed to find out whether these effects are direct or indirect effects. doi:10.1016/j.toxlet.2008.06.428
V38 ECVAM activities in the validation of alternatives for endocrine disruptor testing Miriam Jacobs ∗ , Susanne Bremer, Thomas Hartung, Patricia Pazos, Cristian Pellizzer ECVAM, IHCP EC JRC, Ispra, Italy Legislation or draft proposals in for instance Europe, Japan and the USA require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances and may be putative endocrine disruptors (EDs). To address these requirements and the need to develop alternatives to in vivo testing as part of a tiered testing strategy, ECVAM is involved in European and international collaborations including the OECD test guideline programme. As part of the EU ReProTect Integrated Project, and under the auspices of the OECD, ECVAM is involved in the management of the (pre)validation of several Estrogen (ER) and Androgen receptor (AR) transactivation and ligand binding in vitro assays. Following the ECVAM modular approach to validation and the OECD Guidance Document 34, these tests are have been assessed for modules 1–4, test definition, within laboratory definition, transferability, and between laboratory variability. Most are now are entering module 5 to assess predictive capacity. In addition ECVAM is collaborating with ICCVAM and JaCVAM in the validation of the LUMI cell ER transactivation assays, and with JaCVAM for the validation of the antagonist HeLa ER in vitro assay. doi:10.1016/j.toxlet.2008.06.429 V39 Modeling inflammation–drug interactions lymphocytes–hepatocyte co-culture system
in
vitro:
A
Yong Pil Hwang, Eun Hee Han, Hye Gwang Jeong ∗ Department of Pharmacy, Chosun University, Gwangju, Republic of Korea Xenobiotic–inflammation interactions lead to hepatotoxicity in vivo. The liver immune system comprises a major component of the innate immune system. Lymphocytes are the major cells of the innate immune response; by producing cytokines/chemokines and cytotoxic lysis of transformed cells. Selected xenobiotic agents (acetaminophen, APAP; concanavalin A, Con A; fumonisin B1, FB1) for which this occurs were evaluated for ability to elicit the release of lymphocytes-derived inflammatory mediators. In the current study, cellular interactions between lymphocytes (LCs) and hepatocytes (HPCs) in xenobiotics-induced hepatotoxicity were investigated, using co-cultures of mouse splenic lymphocytes and human hepatoma HepG2 cells. Treatment of the co-cultures with xenobiotics produced hepatotoxicity, whereas either LCs or HPCs cultures alone showed no response to xenobiotics. Expression of TNF-alpha and interferon-gamma was increased in co-cultures but not in the individual cell cultures. These results indicated that LCs and HPCs interact in response to xenobiotics and xenobiotics potentiates the proapoptotic cytokines production in LCs, which may have implications in making HPCs responsive to cytotoxicity of xenobiotics in the liver. In summary, results from these studies using co-cultures of LCs and HPCs qualitatively mirrored data from whole animal studies. Accordingly, this model might have particular utility for assessing drug-inflammation interactions as they relate to inflammatory mediator production. Moreover, this system is amenable to exploration of mechanisms of xenobiotic–inflammation interaction.