Expression And Regulation Of Cytochrome P450 Enzymes And The Related Drug Metabolism In Skin

The skin is a natural barrier between our body and the surroundings, which can protect us from the noxious substances. It also serves as one of the most easily accessible routes of drug administration. The skin is one of the main extrahepatic metabolic organ and expresses many kinds of metabolic enzymes. Cytochrome P450 enzymes (CYP) is a superfamily of heme-containing enzymes. It plays a role in metabolizing both endogenous substances such as steroid, fatty acid, cholic acid, vitamin D3, prostaglandin, catecholamine and exogenous substances such as medicines, pesticides, toxicants, carcinogens. The majority of CYP responsible for the drug metabolism and biotransformation belong to families of CYP1, CYP2, CYP3. They are very important in maintaining normal function of skin and the stability of internal environment. Studying in the expression features of skin CYP is conducive to determine the mechanism of drug transdermal absorption and drug metabolic process in skin, therefore, it is of important significance in increasing the drug effects, developing new percutaneous medicine and preventing adverse reactions of drugs or toxicants on skin.In recent years, a lot of studies on CYP enzymes have proceeded in the area of dermatopharmacology. On one hand, the skin is easy to be inflamed and is hypersensitive, of which the mechanism is complicated. Keratinocyte and fibroblast are involved in the hypersensitiveness, inflamation and immune responses of the skin. The probe of pathological effect in the expression and regulation of CYP enzymes in skin creates a new path for illuminating the mechanism of skin disease and providing the new target for clinical therapy. On the other hand , the therapy of dermatosis always refers to the medicines that are substrates of various kinds of CYP enzymes. The investigation of related metabolism is helpful to rational use of drugs and reducing the adverse reactions.Immunohistochemical and Realtime PCR methods were established in this study to detect the expressions of skin CYP enzymes and to analyze the expression features of keratinocyte and human skin tissue. Oligo GEArray Human Toxicology & Drug Resistance Microarray were applied to the changes of expressions in the lesions of psoriatic patientand the expressions of CYPmRNA in HaCaT cells following histamine stimulating. Using Realtime PCR method to measure the expression and regulation of CYP enzymes in human keratinocyte , to investigate the effects of cytokines (TNF a ,IL-2, IL-6, IFN- Y ) on the expressions of CYP enzymes and the induction of dexamethasone and phenobarbital on CYP. Taking dextromethorphan as CYP2D6's probe drug, the quantities of dextrorphan, a metabolite of dextromethorphan, in rat skin and liver homogenates were measured by LC/MS/MS method and its metabolic process in rat skin homogenate was investigated. CYP2D6 activity and the inhibition of terbinafine on CYP2D6 in rat skin were studied. Part I Using immunohistochemical method to detect the expressions of CYP enzymes in HaCaT cell and human skin tissueImmunohistochemical methods were used to detect the expressions of five enzymes (CYPlAl, CYP1A2, CYP2C9/19, CYP2D6, CYP3A4 ) in CYP1, 2, 3 families which are related to drug metabolism in HaCaT cell and healthy human skin tissue. The results illustrated that:1 There are brown positive expressions of all 5 enzymes in healthy human skin tissue, but the expression strength of the 5 CYP enzymes are different. The expressions of CYP2C9/19, CYP2D6, and CYP3A4 are intensive. CYPlAl is less intensive and CYP1A2 is weak. There are intense expressions of CYP enzymes in skin appendages such as sebaceous glands, hair follicles ,eccrine gland, lymphocyte and monocyte cells.2 There are brown positive expressions in HaCaT cells as well as in liver L02 cells, and the expressions are higher in L02 cell. There are also differences in the intensities of expression in CYP enzymes in HaCaT cells. The expression of CYPlAl is intensive, and the others' are weak. There are no apparent differences among the 5 types of CYP enzymes in L02 cells.Part II Realtime PCR method was used to measure the expression of CYPmRNA in HaCaT cell and human skin tissueRealtime PCR method was established to measure the mRNA expressions of eight CYP enzymes namely CYPlAl, CYP1A2, CYP1B1, CYP2C9, CYPC19, CYP2E1, CYP2D6, CYP3A4, and this method was applied to detect the CYPmRNA in HaCaT cells and healthy human skin tissue as well. The results suggest that:1. CYP1A1, CYP1A2, CYP1B1, CYP2C9, CYP2C19, CYP2E1, CYP2D6 and CYP3A4 can all be detected in healthy human skin tissue, and among these, the expression levels of CYP1A1, CYP1B1, CYP2C19, CYP2E1, CYP2D6 are high, while the expression levels of CYP3A2 and CYP3A4 are low.2. There are significant individual differences among the results of CYPsmRNA expressions in foreskin. The individual difference in CYP1A1 expressions is most conspicuous (RSD=20.12%), and the maximum expression is 115.36 times to the minimum one.When compared with the skins from other parts of the body, the foreskin showed significant differences in CYPmRNA expressions in CYP2C9, CYP2C19, CYP2E1, CYP3A4 (P<0.05), and no significant differences in CYP1A1 and CYP2D6(P>0.05) .When ompared with the trunk and limb skin, pate skin showed significant difference in the expression of CYP1A1 mRNA, and no significant differences in the expressions of CYP1A2, CYP2C9, CYP2C19, CYP2E1, CYP2D6 and CYP3A4mRNA(P>0.05).3. The general expression levels of CYPmRNA in HaCaT cells is consistent with that in healthy human skin tissue, except that the expressions of CYP1A2 and CYP3A4 in HaCaT cells are lower than those in human skin. Part HI Testing CYP450mRNA in psoriatic skin by arrayOligo GEArray? Human Toxicology & Drug Resistance Microarray was used to detect the expressions of 23 CYP enzymes in psoriatic skin and healthy skin. The results have showed that the mRNA expressions of CYP1A1, CYP1A2, CYP1B1, CYP3A5, CYP7B1 in psoriatic skin are 22.99%-32.76% up-regulated, while expressions of CYP24A1, CYP26B1, CYP2A6, CYP2C6, CYP2C9, CYP2D6 and CYP2E1 in psoriatic skin are 21.77%-45.08% down-regulated. The results suggested that there are changes in the expressions of CYP enzymes in psoriatic skin, which may be related to the pathogenesis of psoriasis and immune environment changes in the local impaired skin. In comparison with the healthy subject, the level of NOS in psoriatic skin is about 2.67 folds of that in healthy skin. NO is probably taking part in the regulation of the CYP expressions. Part IV Testing CYPmRNA in histamine stimulated HaCaT cells by arrayIn the study, an in vitro inflammatory skin model was established by stimulating HaCaT cells with histamine, and Oligo GEArray? Human Toxicology & Drug ResistanceMicroarray was used to detect the expressions of CYPmRNA. Through comparing with the untreated control group and Hi receptor antagonist (Cetirizine) intervention group, the effects of histamine on the expressions of CYP enzymes were investigated, and a new path to probe the mechanism and clinical treatment of hypersensitiveness was acquired. The results illustrate that histamine can up-regulate the expressions of CYP4B (22.60%), and the increase can be reversed by Cetirizine. While the expressions of CYP11A1, CYP11B2, CYP1A1, CYP1B1, CYP20A1, CYP26B1, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP3A5, CYP4A11 and CYP4F3 were decreased in the extent of 20.91%-52.30% by histamine. Cetirizine is a new Hi receptor antagonist and is widely used in the treatment of allergic diseases. Cetirizine can inhibit the effects of histamine on CYP expressions in different extents, it indicates that Hi receptor plays a role in the regulation of CYP. As Cetirizine can not completely reverses the decrease of CYP expressions, it is supposed that Hj receptor is not the only way by which histamine affects the CYPmRNA expressions, and there may exist other regulatory mechanisms. Part V Regulations of CYP expressions in human keratinocytesRealtime PCR method was used to measure CYP enzymes' expressions and regulation in human keratinocytes. The effects of cytokines (TNF a , IL-2, IL-6, IFN Y) on the expressions of CYP enzymes as well as the inductions of dexamethasone to CYP3A4, phenobarbital to CYP2C9 and CYP2C19 were investigated. The results suggest that TNF a , IL-6 and IFN Y have no effects on the expressions of CYP2D6 and CYP2El(p>0.05). The Revised CT value of the cells with 20ng/ml, lOOng/ml IL-2 and the controlled cells are 11.86±0.53, 11.62±0.32, 10.53±0.14 respectively, and the differences between the treated cells and controlled cells are significant(p<0.05). At the concentrations of 20ng/ml and lOOng/ml, IL-2 can markedly reduce the expressions of CYP2D6mRNA to 2.51 and 2.13 2.67 folds of that in control cells, but it has no effect on CYPlAlmRNA expressions (P>0.05). Consistent with the CYP expressions in liver, dexamethasone can significantly induce the expressions of CYP3A4mRNA in HaCaT cells(p<0.05). At the concentrations of 2X lO^M, 1X 10"5M and 2xlO'5M, the expressions of CYP3A4mRNA were increase to 11.16, 10.63 and 18.63 times as control cells respectively. Phenobarbital in high concentration (5X lO^M) can induce the expression of CYP2C19 in HaCaT cells. Compare to the control, the difference is significant(PO.05).Part VI CYP2D6 and Dextromethorphan metabolism in skinIn this part of the study, dextromethorphan was taken as the probe drug and incubated in liver skin homogenates at 37°C. The LC/MS/MS method was established to detect the formation amount rate of dextrorphan, which is the metabolite of dextromethorphan, thus the activity of CYP2D6 enzyme was observed. The results indicate that CYP2D6 is able to express in both liver and skin homogenates, dextromethorphan can be transformed to dextrorphan by CYP2D6, but the formation amount of dextrorphan is lower in skin homogenate.At the concentrations of 0.5 to 50ug/ml, 1/[S] of dextromethorphan metabolism is linear to 1 /V in both liver and skin homogenates, and the activity of CYP2D6 in skin is markedly lower than that in liver.Terbinafine is a commonly used antifungal agent. It is a competitive inhibitor of CYP2D6 and can reduce the metabolic activity of CYP2D6. Its inhibition rates in rat liver and skin are 30.22+9.37% and 33.19+7.30 %respectively.