Frank K. Jugert

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

CD8+ dermal T cells from a sulphamethoxazole‐induced bullous exanthem proliferate in response to drug‐modified liver microsomes

There is evidence that T lymphocytes play a critical role in the pathogenesis of drug‐induced bullous exanthems. Sulphonamides are known to be among the most frequent aetiological agents in these severe drug‐induced cutaneous hypersensitivity reactions. Several studies indicate that cytochrome P450‐dependent metabolites of sulphonamides act as the nominal allergens. A 70‐year‐old woman with a severe blistering exanthem caused by cotrimoxazole (sulphamethoxazole and trimethoprim) was studied. We employed an in vitro approach to determine whether cytochrome P450‐dependent enzymes activated drug‐specific T lymphocytes from this patient. Immunohistochemical analysis of involved skin revealed a majority of epidermal CD8+ T lymphocytes, whereas the dermal infiltrate was composed of both CD4+ and CD8+ T cells. Dermal T lymphocytes isolated from lesional skin proliferated in response to sulphamethoxazole, but not to trimethoprim, in the presence of autologous mononuclear cells used as antigen‐presenting cells. The antigen‐specific response of sulphamethoxazole‐specific T cells was significantly augmented in the presence of murine liver microsomes with P450‐dependent catalytic activities. Our observations suggest that some cutaneous hypersensitivity reactions to sulphamethoxazole are due to drug‐specific T lymphocytes. Cytochrome P450‐dependent enzymes may play a critical role in the formation of the nominal antigen, which is recognized by antigen‐specific T cells.

Induction and Inhibition of NAD(P)H: Quinone Reductase in Murine and Human Skin

The purpose of this study was to characterize the human cutaneous NAD(P)H: quinone reductase (NQR) activity by known inhibitors of different reductases and to compare it with the murine skin and liver NQR activity. This enzyme plays a major role in the defence of cells against oxygen stress because it inhibits the 1-electron reduction of quinones to semiquinones and their subsequent oxidation to quinones termed as quinone redox cycle. It belongs to the aromatic hydrocarbon-responsive (Ah) battery. This gene battery includes Cyp1a1 (cytochrome P-450 IA1), Cyp1a2 (cytochrome P-450 IA2) and Nmo-1 [NAD(P)H: quinone reductase]. In the skin cytochrome P-450 IA1-dependent activity is about 1-5% compared to the corresponding activity in the liver, whereas NQR has the same activity in skin and liver. NQR was determined in the cytoplasm of murine skin, liver, and human keratinocytes using 2,6-dichlorophenolindophenol as the substrate. The Ah-receptor binding compounds, such as coal tar constituents, or 3-methylcholanthrene induce cytochrome P-450-dependent activities such as aryl hydrocarbon hydroxylase or 7-ethoxyresorufin-O-de-ethylase and NQR, whereas butyl hydroxytoluol, which does not bind to the Ah receptor, induces only NQR. For inhibition studies several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were used. There was a similar pattern of inhibition of the basal and induced activity in all tissues investigated. Pyrazole, progesterone and phenobarbital did not inhibit, whereas dicoumarol, rutin and indomethacin inhibited NQR activity in murine skin and liver as well as in human keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific IgE and IgG serum antibodies to thiamine associated with anaphylactic reaction

A 47-year-old man came to the office of an orthopedist for treatment of humeral epicondylitis. Thirty minutes after a paraepicondylar injection of Supertendin, the patient experienced itching of palms, trunk, and neck. Dizziness occurred for a few minutes, but no further symptoms developed. Two weeks later, he had a second injection of Supertendin at the same site. With a delay of only 5 minutes, the same symptoms developed. In addition, the patient experienced abdominal cramps, angina pectoris, and scotomas. The patient lost consciousness, and arterial hypotension occurred. He recovered with shock therapy and intravenous administration of steroids. Six weeks and 18 months after the incident, the patient underwent allergy testing. All compounds of Supertendin were repetitively prick tested. A strong positive result could only be obtained from the original preparation or the compound thiamine hydrochloride. All prick tests produced negative results in a control group of patients without history of hypersensitivity (n = 10). All compounds with negative results by prick testing were subsequently applied to the patient either orally or subcutaneously. Lidocaine, phenyl mercury, dexamethasone, and cyanocobolamin did not lead to any positive test reaction. Oral administration of 400 mg of thiamine to the patient was well tolerated.

Drug Metabolism and Drug Allergy

There are several evidences that allergic drug reactions are not due to the parent compound but to its metabolites: a) practolol sensitive patients had an enhanced macrophage inhibition factor production in in vitro assays only after a preincubation of the drug with drug metabolizing liver microsomes; b) patients with drug-induced hepatitis had liver-kidney-microsomal (LKM) antibodies binding specifically to cytochrome P 450 isozymes capable to metabolize those drugs, which induced the hepatitis; c) patients with the phenytoin-hypersensitivity-syndrome have an increased risk to develop allergic reactions to Phenytoin, Carbamazepin, sulphonamids and halothan. First results suggest that they suffer from a glutathione-transferase-deficiency; d) in own results we improved the reactivity of the lymphocytes of patients with drug allergic reaction — indicated by positive results in the skin test or oral challenges — in the lymphocyte transformation test by a preincubation with murine liver microsomes, which are able to metabolize these drugs. The recognition of the role of drug metabolism in type B (hypersensitivity) reactions to drugs will not only improve diagnostic in vitro tests, it will enable us to recognize individual risk factors since there is a high polymorphism of drug metabolizing enzymes especially of the cytochrome P450 isozyme system. Some of them are now detectable by restriction fraction length polymorphism (RFLP) analysis and these and other techniques may lead to the possibility to diagnose special risk factors of the patient before a special drug will be administered.

Cutaneous NAD(P)H:Quinone Reductase: A Xenobiotica-Metabolizing Enzyme with Potential Cancer and Oxidation Stress-Protecting Properties

The toxicity of quinones--including certain chemotherapeutic agents such as doxorubicin--have been related to the enzymatic or nonenzymatic formation of the corresponding semiquinones and their subsequent reaction with molecular oxygen yielding superoxide anion radicals by spontaneous regenerating of the quinones. This semiquinone redox cycling is prevented by the NAD(P)H:quinone reductase (NQR; EC 1.6.99.2) because it mediates a 2-electron reduction which results in the formation of hydroquinones instead of semiquinones. Interestingly, inducers of this enzyme such as butylated hydroxytoluene protect against the severe ulceration of accidental infiltration of doxorubicin into the area around the intravenous infusion. Recently, it has been shown that this highly protective enzyme has a very high basal activity in the epidermis which is in the same range as in the liver. The human gene of the NQR is localized on chromosome 16 and has been cloned recently as well as the gene of the murine liver NQR. We determined NQR in the cytoplasma of murine skin, liver, and human keratinocytes using 2,6-dichlorophenol-indophenol as substrate. In order to characterize this enzyme, induction by polycyclic hydrocarbones and inhibition with several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were determined. There was a similar pattern of inhibition of the basal and induced activity in all tissues so far investigated. Pyrazole, progesterone and phenobarbital did not inhibit; however, rutin and indomethacin inhibited dose-dependently. The most potent inhibitor was dicoumarol. These findings suggest that the same enzymatic form is present in liver and skin, and in murine skin and human keratinocytes.

Severe anaphylactic reaction to topical administration of framycetin

themselves might contr ibute to some of the measured IL-5. However , taking into account the in vitro data and the respective t ime courses of serum IL-5 and blood eosinophils, it is likely that most IL-5 or iginated f rom T cells. In teres t ingly, hypereosinophi l ia re lated to IL-5 secret ion by helper T cells was previously demons t ra ted in pos t t raumat ic eosinophilic pleural effusion? This indicates that different t raumat ic events can lead to T-cell activation, IL-5 release, and hypereosinophil ia . Because activated T cells are known to infiltrate human atherosclerotic plaques, it is possible that in our patient they were mobilized and fur ther s t imulated by the catheter izat ion procedure . Whatever the precise mechanism underlying IL-5 secretion by T cells in atheroembolic disease, our observation strongly suggests that T-cell activation constitutes a major determinant in the hematologic and immunologic abnormalities encountered in this disorder.