4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species.

4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which forms 8-hydroxydeoxyguanosine (8OHdG), oxidative damage. To determine whether reactive oxygen species (ROS) are involved in the generation of 8OHdG by 4NQO, we used high-performance liquid chromatography and immunohistochemistry to measure the levels of 8OHdG in normal human fibroblasts treated with 4NQO. The extent of ROS induced by 4NQO was determined by using fluorescent probes to detect ROS, electron paramagnetic resonance spectrometry using a cell-free system, and measurement of intracellular glutathione (GSH) levels. In fibroblasts, 4NQO dose dependently increased 8OHdG levels. Hydrogen peroxide (H2O2) and superoxide were detected in cells treated with 4NQO by using dichlorofluorescin diacetate and hydroethidine, respectively. The addition of catalase to culture medium reduced 8OHdG levels and the intensity of dichlorofluorescin fluorescence, while 4NQO generated hydroxyl radicals in the cell-free system. These findings suggest that 4NQO treatment leads to formation of superoxide, H2O2, and hydroxyl radicals, resulting in the production of a substantial amount of 8OHdG in DNA. Neither the level of 8OHdG nor that of GSH had returned to the basal level 24 h after removal of 4NQO even at a concentration as low as 1 microM. Our results suggest that generation of ROS and depletion of GSH in cells are also important factors for the generation of 8OHdG by 4NQO. This paper describes practical and sensitive ways to detect ROS and 8OHdG and discusses a new functional pathway to elicit genotoxicity.

Human metallothionein gene expression is upregulated by beta-thujaplicin: possible involvement of protein kinase C and reactive oxygen species.

Recently, we discovered that beta-thujaplicin (BT) induces metallothionein (MT) expression in mouse keratinocytes, both in vivo and in vitro. However, the molecular mechanisms by which BT exerts its biological effects have not been elucidated. The purpose of this study is to explore the signal transduction pathway involved in the MT mRNA induction by BT. Using a HaCaT keratinocyte cell line, Northern blotting was performed for analyzing the human MT-IIA mRNA expression levels in combination with BT and a number of protein kinase (PK) inhibitors including H7, HA1004 and a PKC-specific inhibitor chelerythrin. CAT assays with the MT-IIA gene promorter-CAT construct were conducted for examining the transcriptional regulation by BT of MT. A free radical scavenger N-acetylcysteine (NAC) was used for analyzing a role of oxidative stress for the MT gene induction by BT. BT increased MT-IIA gene transcript levels and CAT activity in a dose-dependent fashion in HaCaT cells. The increase in MT-IIA mRNA levels and CAT activity were completely suppressed by H7 but not by HA1004. In addition, chelerythrin prevented BT-inducible MT-IIA promoter activation. Furthermore, NAC suppressed BT-inducible MT-IIA promoter activation. These results demonstrate that BT is a potent activator of the MT-IIA gene promoter and that PKC activation and reactive oxygen species are implicated in BT-inducible MT-IIA gene expression. BT may be a useful tool for dissecting the signal transduction pathway mediating MT-IIA promoter activation.

Elastin gene expression in blepharochalasis.

Blepharochalasis is a rare condition characterized by recurrent episodes of eyelid edema lead to an atrophic eyelid skin with fine wrinkles and peculiar bronze discoloration. A 32-year-old female presented with loose and redundant skin of the bilateral eyelids. We diagnosed her disease as blepharochalasis by clinical features and by disappearance of elastic fibers from the dermis in the biopsied specimen. Because elastic fibers diminish in the late phase of blepharochalasis, we performed RT-PCR to analyze the mRNA expression of elastin, a major component of elastic fiber. Elastin mRNA expression in the patient's cultured fibroblasts had not decreased compared with that in the control fibroblasts. This result suggests that environmental factors or other matrix components of elastic fibers may be involved in the loss of elastic fiber.

Antibacterial effect of beta-thujaplicin on staphylococci isolated from atopic dermatitis: relationship between changes in the number of viable bacterial cells and clinical improvement in an eczematous lesion of atopic dermatitis.

Beta-thujaplicin (hinokitiol) is a tropolone-related compound purified from the wood of Chamaecyparis obtusa, SIEB: et Zucc. and Thuja plicata D. Don. All Staphylococcus aureus isolates were inhibited by beta-thujaplicin with MICs of 1.56-3.13 mg/L. However, a paradoxical zone phenomenon occurred, with each isolate producing regrowth at higher beta-thujaplicin concentrations. Other antimicrobial agents showed a wide range of MICs. The combination of beta-thujaplicin and zinc oxide inhibited the paradoxical zone phenomenon, and enhanced killing activity against clinically isolated staphylococci. Large numbers of viable bacterial cells, especially S. aureus cells, were detected in the skin surface of atopic dermatitis, in comparison with those in healthy volunteers. The number of cells increased as the severity of the skin condition worsened. Topical application of beta-thujaplicin resulted in a reduction in the number of bacterial cells on the skin surface, and an improvement in skin condition after treatment. The results of this study suggest that the degree of reduction in the number of viable bacterial cells in an eczematous lesion of atopic dermatitis is related to the degree of improvement in skin condition.

Photoaging and oxidative stress.

Photoaging is significantly different from chronological aging in both clinical and histological appearance. It has been suggested that oxidative stress, generated by ultraviolet radiation (UVR), leads to photoaging over a long period. The presence of 8-OHdG, and oxidatively modified proteins such as 4-hydroxy-2-nonenal-modified protein, 3-L-nitro-tyrosine and N(-epsilon) (carboxymethyl)lysine in UV-exposed skin specimens, supports this theory. The pathophysiology of photoaging of the skin caused by chronic inflammation after UVR is reviewed and discussed, with a focus on oxidative stress.

Tissue adhesive and postoperative allergic meningitis.

Adhesives containing cyanoacrylate are used in many surgical procedures, including closure of skin wounds and reinforcement of intracranial aneurysm repairs. A 61-year-old woman developed fever, neck stiffness, and cerebrospinal fluid eosinophilia after an operation for an intracranial aneurysm. We suspected allergic meningitis due to a foreign substance. To detect the cause of this disorder, we did 2-day closed patch testing with the substances thought to be possible allergens. We recorded a positive reaction to methyl-2-cyanoacrylate, the main ingredient of Biobond, the tissue adhesive used in the surgery. This substance could have caused postoperative allergic meningitis.