Skin sensitization resulting for allergic contact dermatitis (ACD) is an occupational and environmental health issue. The allergic hazard for workers and consumers is a serious problem for individuals, employers and marketing certain products. Consequently, it is necessary to accurately identify chemicals skin sensitization potential. According to the new EU chemical regulation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), information of skin sensitization of chemicals manufactured or imported at or above 1 ton/year should be available. Currently, valid approaches assessing skin sensitization rely on animal testing, such as local lymph node assay (LLNA). However, it now ultimately eliminates using animals for this purpose. Based on the fact that a key step in the skin sensitization process is formatting a covalent adduct between allergic sensitizers and proteins and/or peptides in skin, a lot of additional approaches are proposed and developed for replacing or reducing animal used. In this research, three bioassays, 24 h growth inhibition toward Tetrahymena pyriformis, long term (24 h) and short term (30 min) bacterial toxicity (to Vibrio fischeri), and a kinetic glutathione chemoassay are applied for predicting the organic chemicals’ skin sensitization potential. The major results and conclusions obtained are listed as follows: 1. Toxicity enhancement (Te) of 55 chemicals comprising different sensitization potencies were determined and compared with their narcotic toxicity to predict their skin sensitization. Three linear regressions yielded for all allergic sensitizer without nonsensitizers for each bioassay. The linear regressions are improved after classifying sensitizers into five different reaction mechanistic domains. Correspondingly, five different slopes from various reaction mechanisms indicate a decreased sensitivity of toxicity enhancement to skin sensitization potential with order SNAr > SN2 > acylation ≈ Schiff base > aromatic Michael addition. Based on the fact that a key step in the skin sensitization process is forming a covalent adduct between allergic sensitizers and proteins and/or peptides, Te > 10 as a threshold is applied to discriminate these allergic sensitizers, with 100% accuracy for strong (with extreme) and weaker sensitizers, up to 72% accuracy for moderate sensitizers and less than 69% accuracy for nonsensitizers. Compared with these bioassays, a decreasing order of sensitivities is 24 h growth inhibition (Tetrahymena pyriformis) > 24 h growth inhibition (Vibrio fischeri) > 30 min bioluminescence inhibition (Vibrio fischeri). These three bioassays are useful tools for screening sensitization potency of allergic chemicals, and the toxicity enhancement (Te) can be used to discriminate sensitizers from weak or nonsensitizers. However, in this context we should separate aromatic from aliphatic Mas (Michael acceptors). Moreover, metabolic biotransformation should be considered during predicting nonsensitizers’ skin sensitization. 2. Chemical reactivity of selected 55 compounds measuring through kinetic glutathione chemoassay applies to predict their skin sensitization. This chemoassay confirms the fact that the key step of sensitizers eliciting skin sensitization is formatting a covalent adduct between sensitizers and skin proteins or peptides. The chemical reactivity of tested sensitizers strongly relates with their sensitization potential, with strong (extreme) sensitizers presenting the highest reactivity as followed with moderate sensitizers, weak sensitizers as well as nonsensitizers. Moreover, an integrated platform of this chemoassay data and three bioassays data is performed, and this performance shows good sensitivity for monitoring skin sensitization potency, with more rational accuracy for each sensitizing classifications. 3. Thiol reactivity (kGSH) as well as toxicity enhancement (Te) of additional 21 aliphatic α,β-unsaturated compounds are determined for predicting their skin sensitization potential. The linear regressions of skin sensitization versus thiol reactivity and skin sensitization versus toxicity enhancement are significantly improved after classifying these 21 compounds to four chemical subgroups (acrylates, other esters, ketones and aldehydes). Thiol reactivity of these subgroups presented different sensitivity to skin sensitization, with a decreasing order as acrylates (－2.05) > other esters (－1.26) > ketones (－0.43) > aldehydes (－0.21). Moreover, thiol reactivity is confirmed to be a more sensitive tool for predicting skin sensitization, compared with toxicity enhancement. Although the datasets are probably too small to give a definite decision, hydrophobicity reveals contribution to skin sensitization for aliphatic MAs, which is different with literature report. This study suggests that aliphatic MAs should be treated separately into different chemical subgroups for analysis, and their skin sensitization potency can be predicted using kinetic glutathione chemoassay as well as toxicity enhancement bioassay.

ABCB1, a member of the ATP binding cassette (ABC) transporter family, hydrolyses ATP as energy source for the translocation of substrate chemicals across the cell membrane. ABCB1-like transporters are found in all studied species. Typically, these transporters are abundant in tissues that separate compartments of the body such as the blood-brain barrier. Among the ABC transporters the ABCB1-like transporter proteins are of particular interest because they accept a broad variety of substrates and are therefore able to confer multidrug resistance (MDR) and multixenobiotic resistance (MXR) in wildlife, respectively. Inhibitors of the ABCB1-like transporter function can cause chemosensitisation, i.e. accumulation and increased sensitivity of organisms towards potentially harmful (natural/man-made) ABCB1-like substrate chemicals. In zebrafish (Danio rerio) Abcb4 was identified as functionally homologous to ABCB1. The aim of this study was to further characterise Danio rerio Abcb4 and to provide a database to approach the question to what extent ABCB1-like transporter related functions/effects are of ecotoxicological relevance. Main objectives are whether and how known ABCB1 ATPase stimulators and inhibitors interact with Abcb4 ATPase activity; to what extent ABCB1 ATPase assay data are transferable to Abcb4 ATPase assay data; and whether and how environmental chemicals interact with Danio rerio Abcb4 ATPase activity. In this study we established a test system – the ATPase assay with recombinant Danio rerio Abcb4 – to study the interaction of chemicals with the ATPase activity of the transporter protein. To relate obtained data to data for the well-known Homo sapiens ABCB1 and because available data for Homo sapiens ABCB1 were not in all cases suitable for a comparison, the ATPase assay with recombinant ABCB1 was adapted accordingly. Chemicals were tested up to concentrations in the range of their water solubilities to modulate basal and stimulator co-treated Abcb4 and/or ABCB1 ATPase activities. ATPase stimulators are often transported substrates. However, lipophilic compounds stimulating the transporter ATPase activity are not or little transported by transporter action. Therefore, experiments revealing whether compounds are translocated by transporters chemical interference with the transporter protein will not be indicated. Chemicals inhibiting the stimulator (here verapamil) co-treated ATPase activity compete with the verapamil to stimulate ATPase activity or are non-competitive inhibitors. When tested individually, these chemicals can be stimulators or inhibitors of basal ATPase activity, or do not interact with basal ATPase activity. ATPase inhibitors mitigate ATPase activity and ABCB1-like transporter mediated translocation of substrate chemicals. Obtained ATPase assay data were analysed with regard to concentrations at half-maximal effects (EC50s) and effect strengths (percent modulation). ATPase assays with recombinant Abcb4 (at 27 °C) are comparable to ABCB1 ATPase assay data obtained at 37 °C. Danio rerio Abcb4 seems less temperature-sensitive than ABCB1. Calculated activation energies for Abcb4 ATPase activities (40.75 kJ/mol for basal ATPase activity) were up to half as high as those for ABCB1 ATPase activities (81.61 kJ/mol for basal ATPase activity). Larger activation energies were previously proposed to be indicative for larger conformational rearrangements and hence possibly smaller rearrangements take place in Abcb4 compared to ABCB1. Known standard modulators of Homo sapiens ABCB1 ATPase activity interacted specifically with Danio rerio Abcb4 ATPase actitiy. The EC50s of the tested chemicals – 16 of 17 tested chemiacals interacted with the ABCB1 and the Abcb4 ATPase activity – ranged from 0.09 to 296 µM for ABCB1 and from 0.14 to 171 µM for Abcb4. Qualitative ATPase assay data for ABCB1, as interaction or not, seems transferable to Danio rerio Abcb4. Furthermore, when aligning amino acid sequences of mammalian ABCB1 transporter proteins and Danio rerio Abcb4 and comparing ABCB1 residues known to bind to (lipophilic) chemicals no obvious hints were found that chemical binding to Abcb4 is certainly different from ABCB1. Twenty-five of 33 studied environmental chemicals modulated the Abcb4 ATPase activity as stimulators and/or inhibitors. Stimulation of basal Abcb4 ATPase activity was lower for environmental chemicals than for known standard modulators. EC50s of environmental chemicals ranged from below 10 to 357 µM. Effects by environmental chemicals on Abcb4 ATPase activity with EC50s close to their water solubilities may be rather unspecific. The results of this work underline that Abcb4 function is of ecotoxicological importance as on the one hand several environmental chemicals were identified to inhibit Abcb4 ATPase activity – likely acting as chemosensitisers, while on the other hand chemicals stimulating basal ATPase activity suggest that these chemicals are possibly transported. A number of environmental chemicals also inhibited the basal Abcb4 ATPase activity. Especially non-transported inhibitors of the basal Abcb4 ATPase activity would be of ecotoxicological relevance as organisms (here Danio rerio) exposed to these chemicals would not be protected by Abcb4 mediated multixenobiotic resistance and were moreover threatened by chemosensitisation. Future studies should systematically elucidate under which circumstances chemicals are apparently net transported by ABCB1-like transporters and relate these findings to concentrations of environmental chemicals and ABCB1-like transporter protein abundance in wildlife.

ABCB1, a member of the ATP binding cassette (ABC) transporter family, hydrolyses ATP as energy source for the translocation of substrate chemicals across the cell membrane. ABCB1-like transporters are found in all studied species. Typically, these transporters are abundant in tissues that separate compartments of the body such as the blood-brain barrier. Among the ABC transporters the ABCB1-like transporter proteins are of particular interest because they accept a broad variety of substrates and are therefore able to confer multidrug resistance (MDR) and multixenobiotic resistance (MXR) in wildlife, respectively. Inhibitors of the ABCB1-like transporter function can cause chemosensitisation, i.e. accumulation and increased sensitivity of organisms towards potentially harmful (natural/man-made) ABCB1-like substrate chemicals. In zebrafish (Danio rerio) Abcb4 was identified as functionally homologous to ABCB1. The aim of this study was to further characterise Danio rerio Abcb4 and to provide a database to approach the question to what extent ABCB1-like transporter related functions/effects are of ecotoxicological relevance. Main objectives are whether and how known ABCB1 ATPase stimulators and inhibitors interact with Abcb4 ATPase activity; to what extent ABCB1 ATPase assay data are transferable to Abcb4 ATPase assay data; and whether and how environmental chemicals interact with Danio rerio Abcb4 ATPase activity. In this study we established a test system – the ATPase assay with recombinant Danio rerio Abcb4 – to study the interaction of chemicals with the ATPase activity of the transporter protein. To relate obtained data to data for the well-known Homo sapiens ABCB1 and because available data for Homo sapiens ABCB1 were not in all cases suitable for a comparison, the ATPase assay with recombinant ABCB1 was adapted accordingly. Chemicals were tested up to concentrations in the range of their water solubilities to modulate basal and stimulator co-treated Abcb4 and/or ABCB1 ATPase activities. ATPase stimulators are often transported substrates. However, lipophilic compounds stimulating the transporter ATPase activity are not or little transported by transporter action. Therefore, experiments revealing whether compounds are translocated by transporters chemical interference with the transporter protein will not be indicated. Chemicals inhibiting the stimulator (here verapamil) co-treated ATPase activity compete with the verapamil to stimulate ATPase activity or are non-competitive inhibitors. When tested individually, these chemicals can be stimulators or inhibitors of basal ATPase activity, or do not interact with basal ATPase activity. ATPase inhibitors mitigate ATPase activity and ABCB1-like transporter mediated translocation of substrate chemicals. Obtained ATPase assay data were analysed with regard to concentrations at half-maximal effects (EC50s) and effect strengths (percent modulation). ATPase assays with recombinant Abcb4 (at 27 °C) are comparable to ABCB1 ATPase assay data obtained at 37 °C. Danio rerio Abcb4 seems less temperature-sensitive than ABCB1. Calculated activation energies for Abcb4 ATPase activities (40.75 kJ/mol for basal ATPase activity) were up to half as high as those for ABCB1 ATPase activities (81.61 kJ/mol for basal ATPase activity). Larger activation energies were previously proposed to be indicative for larger conformational rearrangements and hence possibly smaller rearrangements take place in Abcb4 compared to ABCB1. Known standard modulators of Homo sapiens ABCB1 ATPase activity interacted specifically with Danio rerio Abcb4 ATPase actitiy. The EC50s of the tested chemicals – 16 of 17 tested chemiacals interacted with the ABCB1 and the Abcb4 ATPase activity – ranged from 0.09 to 296 µM for ABCB1 and from 0.14 to 171 µM for Abcb4. Qualitative ATPase assay data for ABCB1, as interaction or not, seems transferable to Danio rerio Abcb4. Furthermore, when aligning amino acid sequences of mammalian ABCB1 transporter proteins and Danio rerio Abcb4 and comparing ABCB1 residues known to bind to (lipophilic) chemicals no obvious hints were found that chemical binding to Abcb4 is certainly different from ABCB1. Twenty-five of 33 studied environmental chemicals modulated the Abcb4 ATPase activity as stimulators and/or inhibitors. Stimulation of basal Abcb4 ATPase activity was lower for environmental chemicals than for known standard modulators. EC50s of environmental chemicals ranged from below 10 to 357 µM. Effects by environmental chemicals on Abcb4 ATPase activity with EC50s close to their water solubilities may be rather unspecific. The results of this work underline that Abcb4 function is of ecotoxicological importance as on the one hand several environmental chemicals were identified to inhibit Abcb4 ATPase activity – likely acting as chemosensitisers, while on the other hand chemicals stimulating basal ATPase activity suggest that these chemicals are possibly transported. A number of environmental chemicals also inhibited the basal Abcb4 ATPase activity. Especially non-transported inhibitors of the basal Abcb4 ATPase activity would be of ecotoxicological relevance as organisms (here Danio rerio) exposed to these chemicals would not be protected by Abcb4 mediated multixenobiotic resistance and were moreover threatened by chemosensitisation. Future studies should systematically elucidate under which circumstances chemicals are apparently net transported by ABCB1-like transporters and relate these findings to concentrations of environmental chemicals and ABCB1-like transporter protein abundance in wildlife.

Skin sensitization resulting for allergic contact dermatitis (ACD) is an occupational and environmental health issue. The allergic hazard for workers and consumers is a serious problem for individuals, employers and marketing certain products. Consequently, it is necessary to accurately identify chemicals skin sensitization potential. According to the new EU chemical regulation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), information of skin sensitization of chemicals manufactured or imported at or above 1 ton/year should be available. Currently, valid approaches assessing skin sensitization rely on animal testing, such as local lymph node assay (LLNA). However, it now ultimately eliminates using animals for this purpose. Based on the fact that a key step in the skin sensitization process is formatting a covalent adduct between allergic sensitizers and proteins and/or peptides in skin, a lot of additional approaches are proposed and developed for replacing or reducing animal used. In this research, three bioassays, 24 h growth inhibition toward Tetrahymena pyriformis, long term (24 h) and short term (30 min) bacterial toxicity (to Vibrio fischeri), and a kinetic glutathione chemoassay are applied for predicting the organic chemicals’ skin sensitization potential. The major results and conclusions obtained are listed as follows: 1. Toxicity enhancement (Te) of 55 chemicals comprising different sensitization potencies were determined and compared with their narcotic toxicity to predict their skin sensitization. Three linear regressions yielded for all allergic sensitizer without nonsensitizers for each bioassay. The linear regressions are improved after classifying sensitizers into five different reaction mechanistic domains. Correspondingly, five different slopes from various reaction mechanisms indicate a decreased sensitivity of toxicity enhancement to skin sensitization potential with order SNAr > SN2 > acylation ≈ Schiff base > aromatic Michael addition. Based on the fact that a key step in the skin sensitization process is forming a covalent adduct between allergic sensitizers and proteins and/or peptides, Te > 10 as a threshold is applied to discriminate these allergic sensitizers, with 100% accuracy for strong (with extreme) and weaker sensitizers, up to 72% accuracy for moderate sensitizers and less than 69% accuracy for nonsensitizers. Compared with these bioassays, a decreasing order of sensitivities is 24 h growth inhibition (Tetrahymena pyriformis) > 24 h growth inhibition (Vibrio fischeri) > 30 min bioluminescence inhibition (Vibrio fischeri). These three bioassays are useful tools for screening sensitization potency of allergic chemicals, and the toxicity enhancement (Te) can be used to discriminate sensitizers from weak or nonsensitizers. However, in this context we should separate aromatic from aliphatic Mas (Michael acceptors). Moreover, metabolic biotransformation should be considered during predicting nonsensitizers’ skin sensitization. 2. Chemical reactivity of selected 55 compounds measuring through kinetic glutathione chemoassay applies to predict their skin sensitization. This chemoassay confirms the fact that the key step of sensitizers eliciting skin sensitization is formatting a covalent adduct between sensitizers and skin proteins or peptides. The chemical reactivity of tested sensitizers strongly relates with their sensitization potential, with strong (extreme) sensitizers presenting the highest reactivity as followed with moderate sensitizers, weak sensitizers as well as nonsensitizers. Moreover, an integrated platform of this chemoassay data and three bioassays data is performed, and this performance shows good sensitivity for monitoring skin sensitization potency, with more rational accuracy for each sensitizing classifications. 3. Thiol reactivity (kGSH) as well as toxicity enhancement (Te) of additional 21 aliphatic α,β-unsaturated compounds are determined for predicting their skin sensitization potential. The linear regressions of skin sensitization versus thiol reactivity and skin sensitization versus toxicity enhancement are significantly improved after classifying these 21 compounds to four chemical subgroups (acrylates, other esters, ketones and aldehydes). Thiol reactivity of these subgroups presented different sensitivity to skin sensitization, with a decreasing order as acrylates (－2.05) > other esters (－1.26) > ketones (－0.43) > aldehydes (－0.21). Moreover, thiol reactivity is confirmed to be a more sensitive tool for predicting skin sensitization, compared with toxicity enhancement. Although the datasets are probably too small to give a definite decision, hydrophobicity reveals contribution to skin sensitization for aliphatic MAs, which is different with literature report. This study suggests that aliphatic MAs should be treated separately into different chemical subgroups for analysis, and their skin sensitization potency can be predicted using kinetic glutathione chemoassay as well as toxicity enhancement bioassay.