| BackgroundPhotoallergy is a serious skin toxic effect,which is a special skin allergic reaction caused by compounds under sunlight condition(mainly UVA irradiation).The clinical symptoms include erythema,papules and blisters in the exposed parts of skin,such as face,neck,arms and legs,accompanied by itching or burning pain.Systemic symptoms may occur in severe cases.The evaluation of photoallergy is an important item in the safety evaluation of skin medication,some systematic administered drugs and cosmetics.The FDA,ICH and NMPA related guidelines require to make evaluation on compounds with potential photoallergy effect.At present,only one method,the guinea pig photoallergy test,is widely accepted as the photoallergy evaluation method in China and abroad.However,many animals are used in this method and it has a long test period,often causes severe damage to animals and does not conform to the principle of animal welfare.Besides,this method lacks objective and quantitative evaluation indexes.In addition,a number of countries and regions have completely banned the animal testing of cosmetics,and banned the sale of cosmetics that have recently been tested on animals.Therefore,it is urgent to develop in vitro evaluation methods for photoallergy of compounds.ObjectiveThe purpose of this study was to improve the established in vitro THP-1 cell based evaluation methods for photoallergy,determine specific evaluation indexes and criteria for photoallergy,and verify the accuracy,specificity,sensitivity and repeatability of the established method.Methods1.Verification of the in vitro photoallergy evaluation method with cell surface markers as evaluation indexesThe THP-1 cells were incubated with 19 kinds of photosensitizers,4 kinds of photoirritants,2 kinds of skin allergens,1 kind of skin irritant and 1 kind of negative compound for 24 h respectively in cell incubators,with 3-4 concentrations for each test article(one concentration for skin irritant and negative compound).The THP-1 cells received UVA irradiation with the intensity of 1.7 mw/cm2 or kept in dark place for 50 minutes.After UVA irradiation,the cell culture fluid was changed and the cells were cultured in incubators for 24 h.Then the expression levels of CD54 and CD86 on the surface of the THP-1 cells were determined by flow cytometry.The percentage of living cells was determined by propidium iodide(PI)staining.CD54 and CD86 were detected on the basis that the cell viability was more than 750%.The percentages of CD54 and CD86 positive cells and the mean fluorescence intensity(MFR)were measured in the irradiated groups and the unirradiated groups.And the relative fluorescence intensity(RFI)of CD54 and CD86 were calculated.(1)Under the condition of non-irradiation:RFI = "Anti-CD54 or CD86 antibody" MFI in the unirradiated group-MFI of the isotype control of this group/"Anti-CD54 or CD86 antibody" MFI in the cell control unirradiated group-MFI of the isotype control of this group(2)Under the condition of irradiation:RFI = "Anti-CD54 or CD86 antibody" MFI in the irradiated group-MFI of the isotype control of this group/Anti-CD54 or CD86 antibody”MFI in the cell control irradiated group-MFI of the isotype control of this groupWe added pre-irradiation treatment group for the four photoirritants and some photosensitizers.The test materials were first added in cell culture medium(with no cells),UVA irradiated for 30 minutes,kept in dark for 15 minutes,and then incubated with the THP-1 cells.The following test procedures were same as other test articles and the expression levels of CD54 and CD86 were detected.The specific evaluation indexes and criteria were determined for the THP-1 cell based in vitro photoallergy evaluation method with cell surface markers as evaluation indexes,and the accuracy,specificity,sensitivity and repeatability of the established method were verified.2.Verification of the in vitro photoallergy evaluation method of with cytokines as evaluation indexesThe test articles used in this part of study were same as those used in the first part.The THP-1 cells were incubated with different types of test articles mentioned above for 24 h respectively,with 3-4 concentrations for each compound(one concentration for skin irritant and negative compound).Then the THP-1 cells received UVA irradiation with the intensity of 1.7 mw/cm2 or kept in dark place for 50 minutes.Five hours post the UVA irradiation,the contents of IL-8 and TNF-a in the supernatant and lysate of the THP-1 cells were detected using Luminex technique in the irradiated group and unirradiated group of each test article.The ratio of IL-8 content and TNF-a content in the irradiated group versus the unirradiated group were calculated.We added pre-irradiation treatment group for the four photoirritants and some photosensitizers.The test materials were first added in cell culture medium(with no cells),UVA irradiated for 30 minutes,kept in dark for 15 minutes,and then incubated with the THP-1 cells.The following test procedures were same as other test articles and the contents of IL-8 and TNF-a were detected.The specific evaluation indexes and criteria were determined for the THP-1 cell based in vitro photoallergy evaluation method with cytokines as evaluation indexes,and the accuracy,specificity,sensitivity and repeatability of the established method were verified.Results1.Verification of the in vitro photoallergy evaluation method with cell surface markers as evaluation indexesAfter incubation of THP-1 cells with various photosensitizers at different concentrations and irradiation by UVA,15 of the 19 photosensitizers caused significant increase in MFI of CD54 in the irradiated group compared with that in the unirradiated group,and the RFI value of CD54 in the irradiation group was above 1.5.The four photoirritants also significantly increased the MFI of CD54 in the THP-1 cells after incubation and UVA irradiation.However,after the treatment of pre-irradiation,the expression of CD54 in the pre-irradiation group was significantly decreased compared with that in the direct irradiation group(without pre-irradiation).Pre-irradiation can identify photoirritants and photosensitizers.In this study,all the photosensitizers and photoirritants did not cause significant changes in CD86 expression in THP-1 cells prior to and after illumination.Skin sensitizers DNCB and CHD significantly increased the MFI of CD54 in THP-1 cells without UVA irradiation,and there were no further increases but slight decreases in CD54 MFI after irradiation compared with unirradiated DNCB and CHD groups.The expression of CD86 in THP-1 cells increased significantly in both unirradiated and irradiated skin sensitizer groups relative to the cell control groups.The characteristics of CD54 and CD86 expression changes induced by skin sensitizers were significantly different from those induced by photosensitizers.Neither skin irritant nor negative compound caused significant changes in the expression of CD54 and CD86 in THP-1 cells in the irradiated group,and the RFI value of CD54 in the irradiated group was less than 1.5.Based on the above results,MFI and RFI of CD54 expressed in THP-1 cells after incubation with test compounds and UVA irradiation were identified as specific indexes of the in vitro photoallergy evaluation method with cell surface markers as evaluation indexes.The criteria for positive judgement of photosensitizers were preliminarily identified as follows:(1)The MFI of CD54 expressed in THP-1 cells increases significantly in UVA irradiated group relative to that in unirradiated group;(2)RFI of CD54 expressed in THP-1 cells in UVA irradiated group?1.5;(3)When the above two criteria are met,the test article should be pre-irradiated.CD54 RFI in the pre-irradiated illumination group is ?1.5 and is not significant different with the CD54 RFI in the directly irradiated group(without pre-irradiation).If the three criteria are met simultaneously,the test article can be judged as a photosensitizer.In results of method validation showed that the accuracy,specificity and sensitivity of the established method was 85.2%,100%and 78.9%respectively.The repeatability of the method was good.2.Verification of the in vitro photoallergy evaluation method with cytokines as evaluation indexesAfter incubation of THP-1 cells with various photosensitizers at different concentrations and irradiation by UVA,,13 of the 19 photosensitizers induced significant increase in IL-8 content in the irradiated group compared with that in the unirradiated group,and the IL-8 content ratio of the irradiated group versus the unirradiated group was above 6.5.Eight of the 19 photosensitizers induced significant increase in the secretion of TNF-a by THP-1 cells in the irradiation group,but the increase amplitude was obviously less than that of IL-8.The four photoirritants also significantly increased the IL-8 content in the THP-1 cells after incubation and UVA irradiation.However,after the treatment of pre-irradiation,the IL-8 content in the pre-irradiation group was significantly decreased compared with that in the direct irradiation group(without pre-irradiation)and the IL-8 content ratio of the pre-irradiation irradiated group versus the unirradiated group was less than 6.5.The skin sensitizers in this study significantly increased the IL-8 content in THP-1 cells without UVA irradiation,and led to further increases in IL-8 content after irradiation compared with unirradiated groups,but the IL-8 content ratio of the irradiated group versus the unirradiated group was less than 6.5.Under irradiation,skin irritant SLS and negative compound(lactic acid)also significantly increased the secretion of IL-8,but the IL-8 content ratio of the irradiated group versus the unirradiated group was less than 6.5.None of the photoirritants,skin sensitizers,skin irritant and negative compound in this study caused significant changes in TNF-?content in THP-1 cells after UVA irradiation.The results of method verification showed that the accuracy,specificity and sensitivity of the established method was 77.8%,100%and 68.4%respectively.The repeatability of the method was good.ConclusionsIn this study,two in vitro THP-1 cell based evaluation methods for photoallergy with CD54 or IL-8 as evaluation index were established respectively,and the criteria for photoallergy judgement of test articles were determined.It was proved that the two in vitro evaluation methods for photoallergy have good accuracy,specificity,sensitivity and repeatability. |
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