bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunohistochemical detection of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ameloblastomas
Authors: Kumamoto Hiroyuki , Yamauchi Kensuke , Yoshida Mitsuhide , Ooya Kiyoshi
Published: March 2003

Background:  To evaluate the roles of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in tumor progression, expression of MMP‐1, ‐2 and ‐9 and TIMP‐1 and ‐2 was analyzed in ameloblastomas as well as tooth germs.

Methods:  Frozen tissue sections of seven tooth germs and 22 ameloblastomas were immunohistochemically examined using anti‐MMP‐1, ‐2 and ‐9 and anti‐TIMP‐1 and ‐2 antibodies.

Results:  MMP‐1, ‐2 and ‐9 and TIMP‐1 and ‐2 were expressed strongly in mesenchymal components of tooth germs, and stromal cells of ameloblastomas. Immunoreactivity for MMP‐9 in stromal cells of ameloblastomas was significantly stronger than in mesenchymal cells of dental follicles and dental papillae. Dental laminae showed weak MMP‐2 expression in six tooth germs, MMP‐9 expression in two tooth germs and TIMP‐1 expression in six tooth germs. Some tumor cells showed weak MMP‐2 expression in 19 ameloblastomas, MMP‐9 expression in four ameloblastomas and TIMP‐1 expression in all cases. TIMP‐2 reactivity was prominently found in basement membrane zones of dental laminae in tooth germs, and tumor cell islands or nests in ameloblastomas.

Conclusion:  Expression of MMPs and TIMPs was considered to be associated with interactions between epithelial cells and mesenchymal components in normal and neoplastic odontogenic tissues; these molecules might play a role in regulation of tumor progression in ameloblastomas as well as regulation of developmental processes in tooth germs.
Detection of Circulating Melanoma Cells by RT‐PCR Amplification of Three Different Melanocyte‐Specific mRNAs in a Mouse Model
Authors: TSUKAMOTO KATSUHIKO , HIRATA SHUJI , OSADA ATSUSHI , KITAMURA REIKO , SHIMADA SHINJI
Published: March 2003

Three different melanocyte‐specific mRNAs are studied as potential markers for circulating melanoma cells in the serum of mice inoculated subcutaneously with B16F10 melanoma cells. These three mRNAs encode tyrosinase, tyrosinase related protein‐2 (TRP‐2) and Pmel17, proteins that are essential for the synthesis of melanin and are expressed specifically in melanocytes. We used reverse‐transcription polymerase chain reaction (RT‐PCR) to detect these three different melanocyte‐specific mRNAs in the sera of B16F10 bearing mice. Since melanocytes would not normally be present in the blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT‐PCR detection of all three mRNAs was highly sensitive and specific. Our in vitro studies show that as few as 10 melanoma cells can be detected in 125 μl blood and that in vivo, melanoma cells can be detected in blood samples from B16F10 melanoma bearing mice. Of these three mRNAs, Pmel17 mRNA is the most sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA and TRP‐2 mRNA. Moreover, this mouse model might be useful for basic research of malignant melanoma patients with haematogenous metastasis.
Making Sense of Melanosome Dynamics in Mouse Melanocytes
Authors: WU XUFENG , HAMMER III JOHN A.
Published: March 2003

Molecular motors drive most if not all organelle movements in Eukaryotic cells. These proteins are thought to bind to the organelle surface and, through the action of their mechanochemical domains, to translocate the organelle along a cytoskeletal track. In the case of the myosin family of molecular motors, the cytoskeletal track is filamentous actin. Microtubules serve as the cytoskeletal track for the kinesins and dyneins. While a considerable amount is known about the motors and tracks responsible for the bi‐directional movement of pigment granules in fish and frog melanophores, relatively little is known about how melanosomes in mammalian melanocytes are transported out the cells dendritic arbor, accumulated at the ends of these dendrites, and transferred to keratinocytes. In this short review, we focus on the use of video microscopy to address these questions in mouse melanocytes, and we describe how an analysis of melanosome dynamics within wild type and dilute melanocytes shaped our thinking regarding the role of an unconventional myosin in melanosome transport and distribution.
Melanosomal Proteins Promote Melanin Polymerization
Authors: WAGH SUSHAMA , RAMAIAH ABBURI , SUBRAMANIAN RAGHUPATHI , GOVINDARAJAN RAMAN
Published: March 2003

In melanocytes, enzymes involved in the generation of melanin monomers are present and active in coated vesicles which are known to be acidic. Melanin polymerization however, occurs only in melanosomes. In vitro, it is not possible to generate melanin at the acidic pH of melanosomes using 3,4‐dihydroxyphenylalanine (DOPA) and tyrosinase alone whereas melanin readily forms at higher pH with these reagents. Dimerization and elongation of the melanin polymer is known to require deprotonation. We have hypothesized that the amino acid side chains of melanosomal proteins act as proton acceptors to initiate polymerization and that the protonated basic groups serve to attract the negatively charged oligomers thus aiding polymerization and binding to proteins. We show that basic model proteins and basic premelanosomal proteins promote polymerization at an acidic pH and that positively charged surfaces allow binding of the growing melanin polymer. With progressive polymerization and exhaustion of the proton abstracting ability of melanosomal proteins, melanosomal pH drops further, which, we argue, is an additional controlling step that limits tyrosinase activity and melanin polymerization.
Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis
Authors: GIBBS SUSAN , MURLI SEEMA , DE BOER GERRIT , MULDER AAT , MOMMAAS A. MIEKE , PONEC MARIA
Published: March 2003

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus.

In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity.

In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.
Effects of the Actin‐Stabilizing Drug, Jasplakinolide, on Pigment Granule Motility in Isolated Retinal Pigment Epithelial (RPE) Cells of Green Sunfish, Lepomis cyanellus
Authors: KING‐SMITH CHRISTINA , BASCIANO PAUL A. , PHAM NGAN B.
Published: March 2003

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin‐disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin‐stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1–1 μM), but the drug halted movement of most pigment granules and stimulated rapid bi‐directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide‐treated cells demonstrated slow, creeping centrifugal movements and more rapid bi‐directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle‐like structures, which contained F‐actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide‐treated cells revealed a more reticulated network of actin compared to antibody‐labeled control cells. These results indicate that jasplakinolide‐induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.
Xanthophore Migration from the Dermis to the Epidermis and Dermal Remodeling During Salamandra salamandra salamandra (L.) Larval Development
Authors: Pederzoli Aurora , Gambarelli Andrea , Restani Cinzia
Published: March 2003

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, β1‐integrin, chondroitin sulfate, E‐cadherin, N‐cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.
The acute effects of smokeless tobacco (snuff) on gingival blood flow in man
Authors: Mavropoulos Antonios , Aars Harald , Brodin Pål
Published: March 2003

Snuff‐induced blood flow responses in the gingiva were evaluated in 22 healthy casual consumers of tobacco. Laser Doppler flowmetry (LDF) was used to measure blood flow simultaneously and continuously on two gingival sites (buccal aspect of the papillae between the upper lateral incisors and canines). In addition, measurements of skin blood flow in the forehead and palmar side of the left thumb were performed. Arterial blood pressure (BP) and heart rate (HR) were also recorded. Unilateral application of commercial snuff (500 mg, 1%) caused a marked and rapid increase in gingival blood flow (GBF) on the exposed side (p<0.001). Blood flow increased also in the contralateral gingiva and forehead skin (p<0.05). Skin blood flow in the thumb showed an insignificant decrease. BP and HR increased. Vascular conductance increased significantly in the snuff‐exposed gingiva but not in the contralateral gingiva or the forehead. Vascular conductance was largely unaffected in the thumb. It is concluded that acute application of snuff, besides giving rise to typical changes in BP and HR, increases GBF in and around the exposed area, probably through activation of sensory nerves and the subsequent release of vasodilatory peptides from their peripheral endings. Blood flow in unexposed gingival and forehead skin may increase probably due to humoral or nervously mediated mechanisms. However, a passive pressure‐induced hyperaemia in the unexposed gingiva and forehead skin can not be excluded.
Phase 1 evaluation of a local delivery device releasing silver ions in periodontal pockets: safety, pharmacokinetics and bioavailability
Authors: Straub A. M. , Suvan J. , Lang N. P. , Mombelli A. , Braman V. , Massaro J. , Friden P. , Tonetti M. S.
Published: March 2003

A new local delivery device (LDD) capable of releasing silver in periodontal pockets has been developed and tested pre‐clinically. Silver has potent antimicrobial effects on Gram‐negative periodontal pathogens with a mean in vitro minimum bactericidal concentration (MBC) ≤0.5 μg/ml. This phase 1 study assessed the safety, pharmacokinetics and bioavailability of silver ions delivered intracrevicularly with a resorbable LDD (PocketGuardTM) in a group of 9 volunteers affected with periodontitis. In each subject, a PLGA/PEG LDD loaded with 12% silver nitrate (w/w) was inserted in each of 4 selected pockets ≥5 mm. Serum, gingival fluid and subgingival plaque samples were evaluated before and at various time points after LDD placement for 21 days. At each time point, the concentration of silver in gingival crevicular fluid (GCF) was quantified with an Inductively Coupled Plasma‐Mass Spectrometer. Subgingival plaque samples were processed for evaluation of total anaerobic and aerobic counts (CFU/ml). The maximum mean silver concentration in GCF was 1493±709 μg/ml (range 589–2245). It decayed exponentially with a half‐life of 7.1±6.1 days (2.7–20.4). Average silver concentrations in excess of 10 μg/ml were detected in each patient for 14 days after LDD placement with the average concentration for all patients in excess of 25 μg/mL at day 21. Total anaerobic counts decreased an average of 1.7±1.9×106 CFU/ml (p=0.0078) from baseline to day 7, indicating that the silver was biologically active. A mild increase in cervical root discoloration was observed at day 21: 0.25±0.31 stain index units. Discoloration that did not resolve spontaneously could be removed at the end of the study with polishing. No systemic effects were observed. It is concluded that local silver concentrations above the MBC in serum were maintained for at least 21 days. A specific microbiologic effect was also observed.
Levels of tissue inhibitor of metalloproteinases‐1 and matrix metalloproteinases‐1 and ‐8 in gingival crevicular fluid following treatment with enamel matrix derivative (EMDOGAIN®)
Authors: Okuda Kazuhiro , Miyazaki Akira , Momose Manabu , Murata Masashi , Nomura Takashi , Kubota Takehiko , Wolff Larry F. , Yoshie Hiromasa
Published: March 2003

The mechanism of enamel matrix derivative (EMD) action on the periodontal wound healing process is not well understood. However, earlier in vitro studies from our laboratory demonstrated that EMD stimulated the proliferation of both periodontal ligament and gingival fibroblast cells. Therefore, the purpose of this study was to further evaluate the effect of EMD on the early wound healing process by assessing the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gingival crevicular fluid (GCF). Sixteen patients, each of whom had one or two pairs of infrabony defects located contralaterally in the same arch, were included in this clinical trial. Thirty‐six infrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at 2, 4 and 12 week follow‐up evaluation visits, GCF was sampled with paper strips. After determination of GCF volume, TIMP‐1, MMP‐1 and MMP‐8 GCF levels were measured by an enzyme‐linked immunosorbent assay. Intragroup analysis: At week 2 following surgery, when compared to baseline all parameters in each study group, except MMP‐1, significantly increased (p<0.05). There were no significant differences between 4 or 12 weeks and baseline in either study group. Intergroup analysis: At 4 weeks after surgery, GCF volume and TIMP‐1 levels showed a significant decrease (p<0.05) in the EMD group, when compared to the placebo group. MMP‐1 levels at weeks 2, 4 and 12, and MMP‐8 levels at weeks 4 and 12 were significantly lower (p<0.05) in the EMD group compared to the placebo group. EMD compared to placebo treated sites demonstrated a more rapid return to baseline levels of TIMP‐1, MMP‐1 and MMP‐8. These findings suggest that treatment with flap surgery and EMD, compared to flap surgery with placebo, accelerated healing at an earlier stage of wound healing following surgery.
The fibrillar structure of the cemento‐dentinal junction in different kinds of human teeth
Authors: Yamamoto T. , Domon T. , Takahashi S. , Islam M. N. , Suzuki R.
Published: March 2003

The cemento‐dentinal junction was examined in human maxillary incisors, canines and premolars by scanning electron microscopy combined with NaOH maceration. The NaOH maceration was used to remove interfibrillar substances and to observe details of the fibrillar architecture. The teeth were half‐sectioned longitudinally, demineralized and macerated for 3–4 days or for 10–14 days. In the 3–4 day‐macerated specimens, longitudinal sections of the cemento‐dentinal junction were examined. In the 10–14 day‐macerated specimens, the cementum was detached and the inner cementum surface facing the cemento‐dentinal junction was examined. Observations suggested that cemental fibrils intermingle with dentinal fibrils only in places at the cemento‐dentinal junction in both acellular and cellular cementum. These structural features were consistent in all kinds of teeth investigated here. Using human molars, we have previously proposed that the adhesion of proteoglycans is a main factor for the cemento‐dentinal attachment and that the fibril intermingling between dentin and cementum is an accessory or secondary factor. The present study suggests that this applies to other kinds of human teeth.
Salivary IgA subclasses and bacteria‐reactive IgA in patients with aggressive periodontitis
Authors: Hägewald S. , Bernimoulin J.‐P. , Köttgen E. , Kage A.
Published: March 2003

The local salivary immunoglobulin A (IgA) response in patients with aggressive periodontitis to oral microorganisms and its role for the pathogenesis has not been determined. This study investigated the hypothesis that aggressive periodontitis patients have impaired oral secretory immunity. Our test group was made‐up of 19 aggressive periodontitis patients and 19 age‐ and gender‐matched periodontally healthy controls. Total IgA, IgA subclass 1, IgA subclass 2 and IgA reactive to Actinobacillus actinomycetemcomitans Y4, Treponema denticola ATCC 35404 and Candida albicans DSM 3454 were determined by enzyme‐linked immunosorbent assay in whole unstimulated and stimulated saliva. A statistically significantly lower concentration and secretion rate of total salivary IgA (P < 0.01) and IgA1 (P < 0.001) was found in the aggressive periodontitis group in resting and stimulated saliva. A decrease of IgA2 (P < 0.05) was seen in resting saliva. Although only minor differences were detected in the concentration and secretion of bacteria‐reactive IgA in both groups, the proportion of bacteria‐reactive IgA from the total IgA was significantly higher (P < 0.01) in the aggressive periodontitis group in all three microorganisms tested. Our results indicate an inhibition of total secretory IgA. In particular an IgA subclass 1‐specific decrease in aggressive periodontitis was noted, while the bacteria‐reactive humoral immune system in saliva was activated. The role of the decrease of IgA1 immunoglobulins in aggressive periodontitis with respect to susceptibility for periodontal diseases has to be elucidated.
Subjective intensity of pain during the treatment of periodontal lesions with the Vector™‐system
Authors: Braun Andreas , Krause Felix , Nolden Rolf , Frentzen Matthias
Published: March 2003

The aim of this study was to measure subjective intensities of pain during the treatment of periodontal lesions with the Vector™‐system when compared to pain occurring during the treatment with conventional methods.

Twenty patients, each of whom had three teeth with comparable periodontal pocket depths, were treated using three different methods: (i) scaling and root planing with hand instruments, (ii) cleaning with a conventional ultrasonic instrument (Siroson™S) and (iii) cleaning with the Vector™‐system. The subjective intensities of pain during the treatment were measured with an intermodal intensity comparison. A visual analog scale was used for the evaluation after the treatment.

The results of the intermodal intensity comparison during treatment showed that the use of the Vector™‐system caused less pain than the cleaning with hand instruments or the conventional ultrasonic system (P < 0.05). The intermodal intensity comparisons of cleaning with hand instruments and cleaning with the conventional ultrasonic system were not significantly different (P > 0.05). These results could be confirmed by the visual analog scale.

Using the Vector™‐system for cleaning periodontal lesions it is possible to reduce pain sensations compared to conventional methods. Using cleaning methods that cause less discomfort and pain, it might be possible to increase the patient's compliance during non‐surgical periodontal therapy and recall.
Lithium Inhibits the Development of Physical Dependence to Clonidine in Mice
Authors: Dehpour Ahmad Reza , Sadr Seyed Shahabeddin , Azizi Mahtab Roohi , Namiranian Khodadad , Farahani Mehrdad , Javidan Abbas Nowroozy
Published: March 2003

Abstract: Based on our previous finding that chronic lithium treatment reduced naloxone‐precipitated withdrawal syndrome in morphine‐treated mice, the effect of chronic lithium treatment was evaluated on the development of dependence to clonidine. Dependence was induced by injection of either morphine (50, 50 and 75 mg/kg, intraperitoneally with 3 hr interval for 3 consecutive days), or clonidine (2 mg/kg/day, intraperitoneally for 10 days). Naloxone (4 mg/kg, intraperitoneally) precipitated withdrawal signs in both morphine‐ and clonidine‐treated mice. Yohimbine (5 mg/kg, intraperitoneally) precipitated withdrawal signs in the clonidine‐treated mice, similar to morphine withdrawal signs; but failed to precipitate any significant sign in the morphine‐treated mice. Coadministration of lithium was carried out by adding lithium chloride to drinking water (600 mg/l for 20 days; 10 days before the beginning of clonidine administration and 17 days before the administration of morphine to allow the lithium concentration to reach steady‐state). The results indicated that chronic lithium administration significantly attenuated the withdrawal signs, precipitated either by yohimbine or naloxone, in clonidine‐treated mice. As a conclusion, clonidine withdrawal signs are very similar to opioid withdrawal signs, and lithium is able to prevent the development of physical dependence to clonidine.
Cytochrome P‐450 Enzymes Contributing to Demethylation of Maprotiline in Man
Authors: Brachtendorf Lars , Jetter Alexander , Beckurts K. Tobias , Hölscher Arnulf H. , Fuhr Uwe
Published: March 2003

Abstract: From case reports of patients treated with the tetracyclic antidepressant drug maprotiline, it appears that this drug is subject to polymorphic metabolism. Thus, we studied formation of the major maprotiline metabolite desmethylmaprotiline to identify the human cytochrome P‐450 enzymes (CYP) involved. In incubations with human liver microsomes from two different donors, the substrate maprotiline was used at five different concentrations (5 to 500 μM). For selective inhibition of CYPs, quinidine (0.5–50 μM; CYP2D6), furafylline (0.3–30 μM; CYP1A2), ketoconazole (0.2–20 μM; CYP3A4), mephenytoin (20–200 μM; CYP2C19), chlorzoxazone (1–100 μM; CYP2E1), sulphaphenazole (0.2–100 μM; CYP2C9) and coumarin (0.2–100 μM; CYP2A6) were used. Desmethylmaprotiline concentrations were measured by HPLC, and enzyme kinetic parameters were estimated using extended Michaelis‐Menten equations with non‐linear regression. Relevant inhibition of the desmethylmaprotiline formation rate was observed in incubations with quinidine, furafylline and ketoconazole only. Formation rates of desmethylmaprotiline were consistent with a two enzyme model with a high (KM=71 and 84 μM) and a low (KM=531 and 426 μM) affinity site for maprotiline in the two samples, respectively. The high affinity site was competitively inhibited by quinidine (Ki,c 0.13 and 0.61 μM), the low‐affinity site was non‐competitively inhibited by furafylline (Ki,nc 0.11 and 1.3 μM). Thus it appears that CYP2D6 and CYP1A2 contribute to maprotiline demethylation. Based on the parameters obtained, for plasma concentrations of 1 μM 83% (mean) of desmethylmaprotiline formation in vivo is expected to be mediated by CYP2D6 while 17% only may be attributed to CYP1A2 activity.
Acinar Distribution of Rat Liver Arylamine N‐Acetyltransferase: Effect of Chronic Ethanol and Endotoxin Exposure
Authors: Pronko Pavel S. , Järveläinen Harri A. , Lindros Kai O.
Published: March 2003

Abstract: The expression of most drug‐metabolising enzymes is highest in the perivenous region of the liver, where drug‐induced damage is commonly initiated. Arylamine N‐acetyltransferase plays an important role in activation or detoxification of many drugs, carcinogens, pesticides and other xenobiotics, but its acinar distribution is unknown. In this study we have analysed the activity of N‐acetyltransferase in cell lysates obtained from the periportal or perivenous region by digitonin treatment during in situ liver perfusion. Livers from control animals were compared with rats chronically exposed either to ethanol by liquid diet or to lipopolysaccharide (endotoxin) by intravenous administration. The activity of N‐acetyltransferase in the perivenous region was slightly (+ 20%) higher than in the periportal region. Although chronic ethanol exposure did not change total activity, the acinar distribution was reversed to a higher activity in the periportal region. In contrast, chronic endotoxin significantly increased N‐acetyltransferase activity, but did not affect the acinar distribution. This increase was counteracted by simultaneous ethanol treatment. N‐Acetyltransferase activity in perivenous lysates was significantly reduced after the co‐administration of ethanol and endotoxin compared to that after endotoxin alone. Thus, the perivenous zonation of liver N‐acetyltransferase is moderate compared to other transferases or P450 isozymes, and the cellular capacity for N‐acetylation in the perivenous region, where xenobiotic activation to reactive intermediates dominates, may be insufficient.
Drug‐Induced Ventricular Tachyarrhythmia in Isolated Rabbit Hearts with Atrioventricular Block
Authors: Kii Yoshihide , Ito Tsugutaka
Published: March 2003

Abstract: The aims of this study were to develop a suitable model to study proarrhythmic potential using isolated rabbit hearts with atrioventricular block and to examine the proarrhythmic potential of several drugs using this model. With a normal K/Mg solution (K+=5.7 mM and Mg2+=1 mM), d,l‐sotalol (10 and 30 μM), a class III antiarrhythmic drug, prolonged ventricular repolarization, such as QT intervals and monophasic action potential duration, and induced early after‐depolarization and polymorphic ventricular tachyarrhythmia. Cisapride (0.1 and 0.3 μM), a 5‐HT4 receptor agonist, also prolonged the ventricular repolarization, and induced early after‐depolarization. With a low K/Mg solution (K+=1.5 mM and Mg2+=0.35 mM), d,l‐sotalol at 30 μM and cisapride at 0.3 μM more potently prolonged the ventricular repolarization than with a normal K/Mg solution. Furthermore, the incidence of polymorphic ventricular tachyarrhythmia caused by cisapride at 0.3 μM with a low K/Mg solution was higher than that with a normal K/Mg solution. Mosapride citrate, another 5‐HT4 receptor agonist, at 10 μM prolonged the ventricular repolarization and induced early after‐depolarization with a low K/Mg solution, whereas the drug at 1 and 3 μM did not affect any of the parameters examined. Des‐4‐fluorobenzyl‐mosapride, a metabolite of mosapride citrate, at 10 μM slightly prolonged the ventricular repolarization without inducing early after‐depolarization or ventricular tachyarrhythmia. These results suggest that mosapride citrate and des‐4‐fluorobenzyl‐mosapride have much less proarrhythmic potential than cisapride and that isolated rabbit heart with atrioventricular block, perfused with a low K/Mg solution, is a suitable model for predicting the proarrhythmic potential of drugs.
Protective Effect of Aloe Extract against the Cytotoxicity of 1,4‐Naphthoquinone in Isolated Rat Hepatocytes Involves Modulations in Cellular Thiol Levels
Authors: Norikura Toshio , Kennedy David O. , Nyarko Alexander K. , Kojima Akiko , Matsui‐Yuasa Isao
Published: March 2003

Abstract: Aloe is a familiar ingredient in a wide range of health care and cosmetic products and has been reported to possess various physiological effects, antioxidative, anticarcinogenic, antiinflammatory and laxative. Aloe has also been reported to have an effect on liver function. The cytoprotective effect of aloe extract against 1,4‐naphthoquinone‐induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. After exposure to 1,4‐naphthoquinone (100 μM), a decrease in cell viability measured as >60% lactate dehydrogenase depletion was induced. Cellular glutathione (GSH) and protein‐SH levels were also significantly decreased in a time‐dependent manner. However addition of aloe extract resulted in a dose‐dependent improvement of these effects. This cytoprotective effect of aloe could be attributed to its inhibition of GSH and protein‐SH depletions. The effect of the aloe extracts were also dose‐dependent. Addition of diethyl maleate (1 mM), a cellular glutathione‐depleting agent, to hepatocytes treated with both 1,4‐naphthoquinone and aloe extract, induced depletion of GSH, but did not affect protein‐SH or lactate dehydrogenase. These results suggest that the 1,4‐naphthoquinone‐induced toxicity in rat hepatocytes was inhibited by aloe extract, and that this protective effect was due to the maintenance of cellular thiols, especially protein‐SH.
Watercress has No Importance for the Elimination of Ethanol by CYP2E1 Inhibition
Authors: Desager Jean‐Pierre , Golnez Jean‐Luc , De Buck Charlotte , Horsmans Yves
Published: March 2003

Abstract: Watercress, a cruciferous vegetable, is known to inhibit the metabolism of several CYP2E1 substrates such as paracetamol and chlorzoxazone. Since ethanol and its metabolite, acetaldehyde, are CYP2E1 substrates, the influence of watercress on ethanol and acetaldehyde was investigated in healthy human volunteers. According to a randomized cross‐over design, ethanol and acetaldehyde pharmacokinetic parameters were determined in 9 persons at 3 occasions: without watercress and after watercress ingestion preceding ethanol consumption from 1 or 10.5 hr, respectively. Ethanol tmax occurred significantly later when watercress was ingested 1 hr before ethanol ingestion. Likewise, acetaldehyde Cmax was significantly higher whereas acetaldehyde AUCs were increased by watercress but not significantly. All other ethanol and acetaldehyde pharmacokinetic parameters were similar between the 3 treatments. In healthy volunteers, no major watercress effect was observed on ethanol clearance but a weak inhibiting effect on acetaldehyde metabolism is possible. Ethanol absorption is also delayed by single ingestion of watercress immediately preceding ethanol consumption.
Subchronic Toxicity of Baltic Herring Oil and its Fractions in the Rat II: Clinical Observations and Toxicological Parameters
Authors: Stern Natalia , Öberg Mattias , Casabona Helena , Trossvik Christina , Manzoor Ellu , Johansson Niklas , Lind Monica , Örberg Jan , Ricardo Ricardo , Johansson Anna , Chu Ih , Poon Raymond , Yagminas Al , Brouwer Abraham , Jones Bernt , Håkansson Helen
Published: March 2003

Abstract: This study aimed to increase the knowledge about the toxicity of fish‐derived organohalogen pollutants in mammals. The strategy chosen was to separate organohalogen pollutants derived from Baltic herring (Clupea harengus) fillet, in order to obtain fractions with differing proportions of identified and unidentified halogenated pollutants, and to perform a subchronic toxicity study in rats, essentially according to the OECD guidelines, at three dose levels. Nordic Sea lodda (Mallotus villosus) oil, with low levels of persistent organohalogen pollutants, was used as an additional control diet. The toxicological examination showed that exposure to Baltic herring oil and its fractions at dose levels corresponding to a human intake in the range of 1.6 to 34.4 kg Baltic herring per week resulted in minimal effects. The spectrum of effects was similar to that, which is observed after low‐level exposure to pollutants such as chlorinated dibenzo‐p‐dioxins and dibenzofurans (CDD/F) and chlorinated biphenyls, despite the fact that these contaminants contribute to a minor part of the extractable organically bound chlorine (EOCl). The study confirmed previous findings that induction of hepatic ethoxyresorufin deethylase (EROD) activity takes place at daily intake levels 0.15 ng fish‐derived CDD/F‐TEQs/kg body weight. The study also demonstrated that hepatic vitamin A reduction takes place at somewhat higher daily exposure levels, i.e. 0.16–0.30 ng fish‐derived CDD/F‐TEQs/kg body weight. Halogenated fatty acids, the major component of EOCl, could not be linked to any of the measured effects. From a risk management point of view, the study provides important new information of effect levels for Ah‐receptor mediated responses following low level exposure to organohalogen compounds from a matrix relevant for human exposure.
Accumulated p53 protein and UVA protection level of sunscreens
Authors: Seité S. , Moyal D. , Verdier M.‐P. , Hourseau C. , Fourtanier A.
Published: March 2003

Nuclear p53 expression is a sensitive parameter for the detection of ultraviolet (UV)‐induced skin damage, and it has been used as an endpoint to evaluate the effectiveness of sunscreens. In this study, we compared the protection provided by two sunscreens having identical sun protection factors (SPF) but different UVA protection factors (UVA‐PF) measured by the persistent pigment darkening method (PPD). The SPF of the sunscreens was 7 and the UVA‐PF were respectively 7 and 3. Nuclear p53 protein was quantified in human skin biopsies treated with sunscreens and exposed 8 times to 5 MED of solar simulated radiation (SSR). The results showed that both sunscreens offered only partial protection against the increased expression of nuclear p53 protein induced by repetitive SSR exposures. However, a significantly lower level of p53‐positive cells was found in areas protected with the sunscreen having the higher UVA‐PF compared to the other sunscreen protected areas. In order to verify whether the difference in efficacy of these products was due to the difference in UVA absorption capacity, we quantified epidermal p53 protein accumulation after 8 exposures to either UVA (320–400 nm) or UVA1 (340–400 nm). We showed that as with SSR, repetitive exposures to 12.5 and 25 J/cm2 of UVA or UVA1 induced a significant increase in p53‐positive cells in the human epidermis. These results confirmed that SPF determined on the basis of an acute erythemal reaction does not predict the level of protection against cumulative damage. They also showed that the protection provided by two sunscreens with different UVA protection factors is different (based on nuclear p53 protein accumulation), and that the PPD method can distinguish varying levels of sunscreen efficacy against UVA‐induced cell damage.
Low intensity laser irradiation in the treatment of recalcitrant radiation ulcers in patients with breast cancer – long‐term results of 3 cases
Authors: Schindl A. , Schindl M. , Pernerstorfer‐Schön H. , Mossbacher U. , Schindl L.
Published: March 2003

Radiotherapy can be followed by recalcitrant skin ulcers. As low intensity laser irradiation has been demonstrated to have a beneficial effect on impaired wound healing, we investigated its efficacy and safety in three patients with chronic radiation ulcers. The three patients, previously mastectomized due to breast cancer, with recalcitrant radiation ulcers of the skin were treated with a 30 mW helium‐neon laser (wavelength: 632.8 nm, intensity: 3 mW/cm2, dose: 30 J/cm2) three times weekly. In all patients, complete wound closure was achieved within a period of 7, 5, and 8 weeks. One patient died 6 weeks after laser treatment due to tumor cachexia. Neither of the other patients showed recurrence of radiation ulcers or neoplasm during a follow‐up of 36 months. Low intensity helium‐neon laser irradiation has been shown to be effective in the induction of wound healing in radiotherapy‐induced ulcers in three patients with breast cancer.
Regulation of TNFα production and release in human and mouse keratinocytes and mouse skin after UV‐B irradiation
Authors: Yarosh Daniel , Both Dawn , Kibitel Jeannie , Anderson Cathy , Elmets Craig , Brash Douglas , Brown David
Published: March 2003

TNFα is a primary cytokine responsible for inflammatory and immunosuppressive responses in skin. After UV‐B irradiation of cultured human keratinocytes, we found that TNFα was released into the media, as monitored by ELISA, and was bound to cells, as observed by immunofluorescence microscopy. The release of TNFα into cell culture supernatant during the 24 h after UV‐B irradiation was augmented by the addition of IL‐1α to the cells. Further, we found this secretion was unaffected by rapamycin, and therefore independent of FRAP DNA‐protein kinase mediated signal transduction. However, UV‐B also induced expression of membrane‐bound TNFα, and this was dependent on FRAP signaling. In wild type mice, TNFα bound to skin increased immediately after irradiation, declined at 6 h, and then rose again at 12 h before falling by 24 h. This pattern of induction was confirmed by RT‐PCR of TNFα mRNA message in cultured epidermal cells. Induction of membrane‐bound TNFα was also found in c‐fos gene knockout mice deficient in the AP‐1 transcription factor, suggesting that, although AP‐1 containing c‐fos signaling is required for some UV responses, AP‐1 containing c‐fos is not required for this TNFα activation. However, in homozygous p53 knockout mice the basal level of TNFα bound to the epidermis was greatly elevated without UV irradiation. This level declined and remained constant following irradiation. This implies that p53 directly or indirectly represses TNFα gene expression and that modification of p53 mRNA stability or phosphorylation of p53 protein after UV may be responsible for TNFα induction in the membrane. Overexpression of the immunosuppressive cytokine TNFα in this locale may contribute to the carcinogen‐susceptibility of p53 knockout mice.
Effect of ultraviolet A on ornithine decarboxylase and metallothionein gene expression in mouse skin
Authors: Oguro Takiko , Yoshida Takemi
Published: March 2003

Background: Ultraviolet A (UVA) is known to induce the expression of many stress responsive genes due to the generation of reactive oxygen species (ROS). However, UVA's role in inducing metallothionein (MT) gene expression has not been studied. Furthermore, our group demonstrated that UVA enhanced 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐mediated induction of ornithine decarboxylase (ODC) activity in mouse skin (1).

Methods: We examined the interaction of UVA, TPA and antioxidants on the induction of MT and ODC mRNA in mouse skin. Female CD‐1 mice were exposed to UVA (19 J/cm2) and total RNA was isolated from the skin. Northern blot analysis for MT and ODC mRNAs was performed. ODC activity in mouse epidermis was also determined in some experiments.

Results: UVA induced MT mRNA in mouse skin; however, it did not increase ODC mRNA. 1,4‐Diazabicyclo‐[2,2,2]‐octane (DABCO), a singlet oxygen scavenger, reduced UVA‐mediated induction of MT mRNA by 40%. The data suggest that ROS produced by UVA exposure may contribute to its ability to induce MT mRNA. UVA slightly enhanced TPA‐mediated ODC mRNA induction, while it enhanced ODC enzyme activity 70%. UVA additively intensified TPA‐mediated MT mRNA induction. α‐Tocopherol pretreatment inhibited the induction of ODC enzyme activity by TPA treatment combined with UVA exposure (TPA+UVA); however, α‐tocopherol had less of an inhibitory effect on ODC mRNA induction by TPA+UVA. Curcumin, a plant pigment, dramatically inhibited both TPA‐ and TPA+UVA‐induced expression of ODC and MT genes.

Conclusions: These results demonstrate that UVA can induce MT gene expression and enhance TPA‐induced ODC and MT gene expression. The data further suggest that these effects are partially mediated by ROS.
Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair
Authors: Sutherland Betsy M. , Hacham Haim , Bennett Paula , Sutherland John C. , Moran Michael , Gange R. W.
Published: March 2003

Background/Aims: Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV‐irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin.

Methods and Results: Using a solar simulator filtered with three types of UV‐filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV‐filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair).

Conclusions: Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.
Acute effects of UVB radiation on the proliferation and differentiation of keratinocytes
Authors: Lee J. H. , An H. T. , Chung J. H. , Kim K. H. , Eun H. C. , Cho K. H.
Published: March 2003

Purpose: The effects of UVB radiation on the proliferation and differentiation of epidermal keratinocytes were investigated with respect to timing, dosage, and repeated exposures.

Methods: Nine healthy volunteers were placed into three subgroups and exposed to UVB radiation on buttock skin using a Waldmann UV 800 unit fitted with Philips TL‐20W/12 fluorescent lamps. Three volunteers were given 2 MED of UVB and biopsied at: pre‐exposure, 24, 48 and 72 h after UVB exposure. For three volunteers, 1 MED, 2 MED, 3 MED of UVB were applied. After 48 h, biopsies were taken from non‐irradiated and irradiated sites. Finally, three volunteers received 1 MED of UVB daily for 5 days, and the non‐irradiated and irradiated sites were biopsied 48 h after the final exposure. The expression of proliferation and differentiation markers by keratinocytes were detected by immunohistochemical staining, and the results were analysed quantitatively by image analysis.

Results: The expression of proliferation and differentiation markers was observed prominently 48 h after irradiation. Higher doses of UVB caused an increase in proliferation and differentiation marker expression. Repeated exposures potentiated the effect of UVB radiation.

Conclusion: UVB irradiation concomitantly promotes epidermal proliferation and differentiation. Responses were maximal 48 h after irradiation. This effect of UVB increases linearly according to dose and repetition.
Visual discrimination testing of ultraviolet transmitting and ultraviolet blocking acrylic thermoplastics
Authors: Kucenic Michael J. , Patel Manisha , Feldman Steven R. , Liguori Anthony , Fleischer Alan B.
Published: March 2003

Background: Visual perception has hindered the development of blinded, placebo‐controlled ultraviolet light exposure conditions in humans. New acrylic thermoplastics that block or transmit ultraviolet light have visual properties that may provide a solution.

Methods: In a series of triangle taste tests, 60 subjects were tested for the ability to visually perceive ultraviolet light transmission by two of four types of acrylic thermoplastics.

Results: Forty‐six of sixty (67%) subjects were unable to visually detect ultraviolet light transmitting acrylics among ultraviolet blocking acrylics in both an ambient light background condition and an ambient light background condition with ultraviolet A light supplementation.

Conclusion: Acrylic thermoplastics allow for the production of blinded, placebo‐controlled ultraviolet light exposure conditions by eliminating visual discrimination differences between active and control groups.