Jarmo Laihia

Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines

We have examined the effects of UVB irradiation, oxidative stress and cytokines on the antioxidant enzymes copper/zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in HeLa cells. A single dose of UVB irradiation regulated dose-dependently the expression of the 4 kb transcript of MnSOD although it did not have any significant effect on MnSOD enzymatic activity. In contrast, UVB irradiation reduced both the enzymatic activity and the expression of the 0.7 and 0.9 kb mRNA transcripts of CuZnSOD. The cytokines TNF-alpha (1 ng ml-1 and 10 ng ml-1) and IL-6 (100 U ml-1) induced MnSOD activity, and TNF-alpha also upregulated MnSOD mRNA expression. Interestingly, genistein, a soy isoflavone and a tyrosine kinase inhibitor, was able to inhibit the induction of Mn-SOD activity and mRNA expression by TNF-alpha. Enzymatic CuZnSOD activity was depressed by a high dose of H2O2 while IL-6 or TNF-alpha had no effect on CuZnSOD activity. Our results demonstrate that, in addition to enzyme activity level, UVB irradiation can regulate the superoxide dismutases at the mRNA level. We also suggest that UVB irradiation, oxidative stress and cytokines regulate differentially CuZnSOD and MnSOD, and that the activities and expression of these antioxidant enzymes are controlled by distinct mechanisms.

Lucigenin and linoleate enhanced chemiluminescent assay for superoxide dismutase activity

The xanthine/xanthine oxidase dependent chemiluminescence was enhanced both by lucigenin and linoleate to create a sensitive, specific, and rapid chemiluminescent method for superoxide dismutase (SOD) activity determination. A pH optimum at around 10.0 was found both for the chemiluminescence and its inhibition by SOD. At this pH, a linear inhibition response to concentrations from 0.01 to 100 ng/ml of bovine Cu,Zn SOD could be established, with a 50% inhibitory concentration of 0.75 ng/ml. As little as 0.17 fmol of Cu,Zn SOD per test can be detected. With a slightly lower sensitivity, the method is operative at pH 7.4, too. Both Cu,Zn SOD and Mn SOD can be assayed. The rationale of the assay is in combining a superoxide-producing enzymatic system with linoleate amplification to enhance the sensitivity of the chemiluminescence to inhibition by SOD activity. Applicability of the method to biological samples was tested with a standard addition experiment.

UV-Induced Tolerance to a Contact Allergen Is Impaired in Polymorphic Light Eruption

Polymorphic light eruption (PLE) is a common skin disorder provoked by exposure to UVR. Its clinical symptoms resemble those of a contact allergic reaction. PLE is generally considered a T-cell-mediated autoimmune reaction toward a yet unidentified antigen formed in UVR-exposed skin. Predisposition to such an immune reaction may result from aberrant epitope formation, increased immune reactivity to a universal epitope, or diminished propensity to UVR-induced immunosuppression or to the induction of tolerance. In a study comprising a total of 24 PLE patients and 24 healthy sex- and age-matched controls, we found that both groups demonstrated similar immunosuppression of contact sensitization to diphenylcyclopropenone by earlier exposure to solar-simulating UVR. However, only 1 out of 13 PLE patients (8%) versus 6 out of 11 controls (55%) that had been immunosuppressed by UVR exhibited a state of immunotolerance toward the same allergen after 10-24 months (P=0.023). We conclude that the impaired propensity to UVR-induced allergen-specific immunotolerance may promote recurrent PLE.

Protodynamic Intracellular Acidification by cis-Urocanic Acid Promotes Apoptosis of Melanoma Cells In Vitro and In Vivo

The extracellular tumor microenvironment is acidified, whereas the intracellular pH of tumor and stromal cells is neutral. cis-Urocanic acid (cis-UCA), an endogenous compound of the skin, can acidify the cytosol by transporting protons into the cells. This phenomenon, termed the protodynamic concept, was studied here in human cancer cells. cis-UCA dose-dependently reduced the number of viable human melanoma, cervical carcinoma, and fibrosarcoma cells at weakly acidic extracellular pH. The intracellular pH decreased by up to 0.5 pH units in a concentration-dependent manner with 0.3-30  m cis-UCA at extracellular pH 6.5 but not at pH 7.4. Under the same conditions, 30  mM cis-UCA induced annexin-V binding and activation of caspase-3 in A2058 melanoma cells as signs of apoptotic cell death. Finally, growth of human melanoma xenografts in SCID mice was suppressed by 60% following intratumoral injection of cis-UCA. Accordingly, the percentage of tumor necrosis and active caspase-3-immunopositive cells increased, whereas proliferation activity decreased. These results identify cis-UCA as an anticancer agent inhibiting melanoma growth by immediate intracellular acidification followed by apoptotic cell death in vivo.

Three randomised phase I/IIa trials of 5% cis-urocanic acid emulsion cream in healthy adult subjects and in patients with atopic dermatitis.

New treatment modalities are needed in atopic dermatitis. We evaluated the pharmacokinetics, safety, tolerability, and efficacy of topical cis-urocanic acid (cis-UCA) cream in randomised vehicle-controlled double-blinded clinical trials. The subjects received 5% cis-UCA emulsion cream and control vehicle on volar forearms after right-left randomisation. Study 1: 16 healthy subjects received one dose on the skin and, a week later, on DMSO-irritated skin. Study 2: 16 healthy subjects received 2 daily doses for 10 days. Study 3: 13 patients with mild to moderate disease were treated on selected skin lesions twice daily for 28 days. Study treatments were well tolerated. cis-UCA remained close to endogenous levels in plasma and urine. cis-UCA reduced transepidermal water loss (TEWL) both in healthy subjects and in the patients. Eczema area severity index and physicians global assessment improved from baseline with both treatments. cis-UCA cream improved skin barrier function and suppressed inflammation in the human skin.

Establishment of a kinetic model for urocanic acid photoisomerization

Abstract The photoisomerization reactions of the E and Z isomers of urocanic acid (UCA), which are natural constituents of the mammalian epidermis, were studies by time-resolved flash photolysis and quantum yield determination in the pH range 3.0–10.0. A kinetic model for the processes is presented on the basis of the following observations: (1) the sum of the quantum yields for E → Z and Z → E reactions can be more than unity; (2) a kinetic component with almost equal rate parameters for both reactions is observed in flash photolysis; (3) fluorescence and (4) loss of material were not detected; (5) the quantum yield of formation of the isomerization product is represented by a short-lived decay component in the E → Z and by a long-lived component in the Z → E reaction. Correlations between the experimental data and possible molecular arrangements are examined.

Effects of intramammary infusion of cis–urocanic acid on mastitis-associated inflammation and tissue injury in dairy cows

OBJECTIVE To evaluate the effects of cis-urocanic acid (cis-UCA) on mammary gland (MG) inflammation and injury associated with Escherichia coli-induced mastitis in dairy cows. ANIMALS 12 lactating dairy cows (36 MGs). PROCEDURES At 7-week intervals, a different MG in each cow was experimentally inoculated with E coli. At 6-hour intervals from 6 to 36 hours after inoculation, the inoculated MG in each cow was infused with 40 mL of saline (0.9% NaCl) solution, 12.5mM cis-UCA, or 25mM cis-UCA (4 cows/group); ultimately, each cow received each treatment. Immediately prior to and at various time points after inoculation and treatment, milk samples were collected. Bacterial CFUs, somatic cell counts (SCCs), N-acetyl-beta-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) activities, and concentrations of bovine serum albumin, tumor necrosis factor-alpha, and cis-UCA were quantified in each milk sample. Results-Compared with findings in saline solution-treated MGs, NAGase and LDH activities in milk samples from cis-UCA-treated MGs were lower. Cis-UCA had no effect on milk SCCs and milk concentrations of bovine serum albumin and tumor necrosis factor-alpha. Furthermore, cis-UCA had no adverse effect on bacterial clearance; CFUs of E coli in MGs treated with saline solution or cis-UCA were equivalent. CONCLUSIONS AND CLINICAL RELEVANCE In cows, milk NAGase and LDH activities were both lower in E coli-infected MGs infused with cis-UCA than in those infused with saline solution, which suggests that cis-UCA reduced mastitis-associated tissue damage. Furthermore, these data indicated that therapeutic concentrations of cis-UCA in milk can be achieved via intramammary infusion.

Protodynamic therapy for bladder cancer: in vitro results of a novel treatment concept

To present a novel treatment approach for urinary bladder cancer, protodynamic therapy, which comprises inhibition of cancer cell proliferation by intracellular acidification; cis‐urocanic acid (cis‐UCA) was investigated as a protodynamic drug in bladder cancer cell cultures and compared with conventional chemotherapeutic agents.

Trans‐urocanic acid, a natural epidermal constituent, inhibits human natural killer cell activity in vitro

Abstract UV irradiation has been reported to influence NK cell function both in vitro and in vivo. Since urocanic acid may mediate UV‐induced immune modulation we tested the effect of trans‐ and cis‐urocanic acid (UCA) on the cylotoxic activity of human peripheral blood lymphocytes against the erythroleukemic target cell line K562 in vitro. Trans‐UCA was found to be a strong inhibitor of NK cell activity whereas cis‐UCA had no effect. Trans‐UCA also partially inhibited cytotoxic function of IL‐2‐activated NK cells and reduced IL‐2‐induced activation of NK cells. This is the first report describing trans‐UCA to be active, and cis‐UCA inactive, in regulating an immune function. In the skin, a decrease in epidermal trans‐urocanic acid concentration by UV radiation could produce a favorable milieu for NK cell activity, and thus counteract the impairment of antigen‐specific immune surveillance, induced by increased cis‐urocanic acid concentrations.

Adaptation of the Human Skin by Chronic Solar-simulating UV Irradiation Prevents Ultraviolet-B Irradiation-induced Rise in Serum C–Reactive Protein Levels¶

Exposure of the skin to UV radiation induces local inflammation. We hypothesized that inflammation induced by erythemal UV‐B irradiation could elevate levels of serum C‐reactive protein (CRP) and that suberythemal repeating doses of solar‐simulating UV radiation (SSR) would produce photoadaptation to such inflammation. Separation‐free high‐sensitivity assays of CRP show an increase by 42% (P= 0.046) in CRP concentrations in healthy human subjects 24 h after a 3 minimal erythemal dose (MED) dose of UV‐B delivered onto a 100 cm2 skin area. Preceding daily suberythemal doses of whole‐body SSR for 10 or 30 consecutive days completely prevented the CRP increase. UV‐B‐induced skin erythema was partially attenuated by 30 preceding days of SSR only (P= 0.00066). After 10 daily SSR doses, the mean baseline CRP concentrations (0.24 ± 0.21 mg/L) declined by 35% (P= 0.018). Using high‐sensitivity analysis of serum CRP as the endpoint marker for cutaneous inflammation, we show that acute exposure of even a relatively small skin area to erythemal UV‐B induces skin inflammation detectable also at the systemic level and that photoadaptation by preceding repeating suberythemal doses of SSR reduces signs of inflammation. Our data complement the view given by previous studies in that local photoadaptation also has systemic manifestations.