Study Of Human Cell Line Activation Test (h-CLAT) For Evaluating The Sensitization Potential Of Chemicals

Objective:Using THP-1 cells(human monocyte cell lymphoma) to establish human Cell Line Activation Test(h-CLAT) for the detection of potential contact allergens.To compare the results of h-CLAT with those of the traditional methods including human data,guinea pig test and LLNA test and evaluate the accuracy of h-CLAT to distinct between allergens and non allergens.And to provide more data for the validation of h-CLAT.Method:1.The expression of the co-stimulatory molecules CD86 and CD54 on THP-1 cells after a 24h exposure to DNCB was measured using flow cytometry.Then to study the relationship between CD86/CD54 expression and THP-1 cell viability, and evaluate the appropriate test doses in h-CLAT to distinguish between allergens and non-allergens.2.Five allergens including 2,4-dinitrobenzene(DNCB),potassium dichromate(PD), hydroquinone(HQ),nickel sulfate(Ni),eugenol(EU) and two non-allergens sodium lauryl sulfate(SLS),glycerol(GC) were evaluated using h-CLAT test.The expression of CD86 and CD54 on THP-1 cells and cell viability was measured using flow cytometry.Then to compare the results of h-CLAT with guinea pig test and LLNA,and evaluate the accuracy of h-CLAT to distinct between allergens and non-allergens from the data.Results:1.In this study,the expression of CD86 and CD54 on THP-1 cells and cell viability using flow cytometry after a 24h exposure to DNCB was measured.The results showed that the expression of CD86/CD54 was increased significantly in the cell viability range of 65%to 95%.When CV＞95%.the alteration of CD86/CD54 expression was not observed.The results suggest that cytotoxicity(65%-95%cell viability) was needed for enhancement of CD86/CD54 expression.2.Five known allergens including DNCB,PD.HQ.Ni.EU and two non-allergens SLS,GC were evaluated using h-CLAT test.The results showed that DNCB,PD,HQ, Ni,EU cause a significant increased expression of CD86,and DNCB,PD,HQ,EU cause a significant increased expression of CD54.However the alteration of CD86/CD54 expression was not observed following treatment with non-allergens SLS and GC.From the data,the criterion of an RFI＞=150 for CD86 expression gave a 100%accuracy.And for CD54 expression,when the criterion was an RFI of 150,the accuracy was 85%.It suggested that compared with CD54,CD86 was more sensitive.The results suggested that h-CLAT method using THP-1 cells may be a useful in vitro skin sensitization model to predict various contact allergens.Conclusion:In h-CLAT method,for most allergens cytotoxicity(65%-95%cell viability) was needed for enhancement of CD86/CD54 expression.And the criteria of "CD86≧150 or CD54≧150" resulted in an accuracy above 85%.These findings suggest that h-CLAT method using THP-1 cells would be an useful in vitro skin sensitization model to predict various contact allergens.