Kohji Aoyama

Allergy to laboratory animals: an epidemiological study

A large cross sectional survey was carried out using a self administered questionnaire to examine the prevalence of laboratory animal allergy (LAA) and the factors associated with its development. Out of 5641 workers who were exposed to animals at 137 laboratory animal facilities in Japan, 23.1% had one or more allergic symptoms related to laboratory animals. The commonest symptom as rhinitis. About 70% of LAA subjects developed symptoms during their first three years of exposure. Atopy (past and family history), the number of animal species handled, and the time spent in handling correlated significantly with the development of LAA as did some types of job. A close relation between nasal symptoms and exposure to rabbits and between skin symptoms and exposure to rats were found. LAA subjects developed symptoms most quickly to rabbits.

Method to overcome photoreaction, a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species

Non-fluorescent dichlorofluorescin (DCFH) was converted to fluorescent products by photo-irradiation during observations with spectrofluorometer and fluorescence microscopy. Photo-irradiation of DCFH at 250, 300, 330, 400, 500, or 600 nm generated fluorescent dichlorofluorescein (DCF), an oxidation product of DCFH, and an unrecognized fluorescent product. The ratio of the unknown product to DCF varied from 0.15 to 8.21 depending on wavelength. Although reactive oxygen species scavengers, such as catalase, superoxide dismutase, and sodium azide, did not suppress the increase in non-specified fluorescence, reagents such as ascorbic acid, mercaptopropionyl glycine, and methoxycinnamic acid, in a cell-free system, almost completely suppressed it with little effect on the fluorescence of DCF. Meanwhile, ascorbic acid also suppressed non-specified fluorescence in cells, but not completely. At low concentrations of DCFH, the speed of increasing fluorescence was considerably retarded, to such a degree that the fluorescence increase in cells during fluorescence microscopic observation was negligible. The addition, at the time of evaluation, of the above reagents to cell-free systems and, in cell systems, reducing the concentration of DCFH, effectively suppressed the photoreaction of DCFH.

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.

Effects of benzene inhalation on lymphocyte subpopulations and immune response in mice

To clarify the immunotoxicity of benzene, the effects of benzene inhalation on T and B lymphocytes and immune responses in mice were examined. BALB/c male mice were exposed to 50 or 200 ppm benzene vapor, 6 hr/day for 7 or 14 consecutive days. T and B lymphocytes, in blood and spleen, were detected by the cytotoxicity assay with anti-Thy-1.2 monoclonal antibody and the membrane immunofluorescence test with anti-immunoglobulin antibody, respectively. Humoral immune response to sheep red blood cells was determined by the hemolytic plaque-forming cell assay. Cell-mediated immune response was measured by contact sensitivity (CS) to picryl chloride. The activity of suppressor cells was evaluated in spleen by the suppressive effect on passive transfer of CS. The ratio and absolute number of T and B lymphocytes in blood and spleen were depressed after a 7-day exposure at 50 ppm benzene. The depression of B lymphocytes was dose dependent and more intense than that of T lymphocytes. The ability to form antibodies was suppressed by benzene at all exposure levels, but the CS response was resistant to benzene inhalation and rather enhanced at 200 ppm exposure for 14 days. The activity of suppressor cells could not be detected at this dose level. These data show that benzene inhalation effects on humoral and cell-mediated immune responses are a result of the selective toxicity of benzene to B lymphocytes and suppressor T cells.

Habitual exercise induced resistance to oxidative stress

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15–60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.

P53 plays an important role in cell fate determination after exposure to microcystin-LR.

Background Microcystin-LR, a cyclic heptapeptide, possesses the ability to inhibit the serine/threonine protein phosphatases PP1 and PP2A and, consequently, exhibits acute hepatocytotoxicity. Moreover, microcystin-LR induces cellular proliferation, resulting in tumor-promoting activity in hepatocytes. However, mechanisms that regulate the balance between cell death and proliferation after microcystin-LR treatment remain unclear. Objective We examined the contribution of the transcription factor p53, as well as that of the hepatic uptake transporter for microcystin-LR, organic anion transporting polypeptide 1B3 (OATP1B3), to the cellular response to microcystin-LR exposure. Methods We analyzed intracellular signaling responses to microcystin-LR by immunoblotting and real-time reverse-transcriptase polymerase chain reaction techniques using HEK293 human embryonic kidney cells stably transfected with SLCO1B3 (HEK293-OATP1B3). In addition, we analyzed the effect of attenuation of p53 function, via the p53 inhibitor pifithrin-α, and knockdown of p53 mRNA on the cytotoxicity of microcystin-LR using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results Microcystin-LR induced the phosphorylation and accumulation of p53 in HEK293-OATP1B3 cells, which resulted in up-regulation of the expression of p53 transcript targets, including p21 and seven in absentia homolog 1 (siah-1). In addition, microcystin-LR activated Akt signaling through the phosphorylation of Akt and glycogen synthase kinase 3β. Although Akt signaling was activated, the accumulation of p53 led cells to apoptosis after treatment with 50 nM microcystin-LR for 24 hr. Both pharmacological inhibition of transcription factor activity of p53 by pifithrin-α and knockdown of p53 with small hairpin RNA attenuated the susceptibility of HEK293-OATP1B3 cells to microcystin-LR. Conclusions This study demonstrates the importance of p53 in the regulation of cell fate after exposure to microcystin-LR. Our results suggest that, under conditions of p53 inactivation (including p53 mutation), chronic exposure to low doses of microcystin-LR may lead to cell proliferation through activation of Akt signaling. Results of this study may contribute to the development of chemoprevention and chemotherapeutic approaches to microcystin-LR poisoning.

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung

BackgroundBronchus-associated lymphoid tissue (BALT) is the secondary lymphoid tissue in bronchial mucosa and is involved in the development of bronchopulmonary immune responses. Although migration of lymphocytes from blood vessels into secondary lymphoid tissues is critical for the development of appropriate adaptive immunity, the endothelia and lymphocyte adhesion molecules that recruit specific subsets of lymphocytes into human BALT are not known. The aim of this study was to determine which adhesion molecules are expressed on lymphocytes and high endothelial venules (HEVs) in human BALT.MethodsWe immunostained frozen sections of BALT from lobectomy specimens from 17 patients with lung carcinoma with a panel of monoclonal antibodies to endothelia and lymphocyte adhesion molecules.ResultsSections of BALT showed B cell follicles surrounded by T cells. Most BALT CD4+ T cells had a CD45RO+ memory phenotype. Almost all BALT B cells expressed α4 integrin and L-selectin. In contrast, 43% of BALT T cells expressed α4 integrin and 20% of BALT T cells expressed L-selectin. Almost all BALT lymphocytes expressed LFA-1. HEVs, which support the migration of lymphocytes from the bloodstream into secondary lymphoid tissues, were prominent in BALT. All HEVs expressed peripheral node addressin, most HEVs expressed vascular cell adhesion molecule-1, and no HEVs expressed mucosal addressin cell adhesion molecule-1.ConclusionHuman BALT expresses endothelia and lymphocyte adhesion molecules that may be important in recruiting naive and memory/effector lymphocytes to BALT during protective and pathologic bronchopulmonary immune responses.

Allergic contact dermatitis in shiitake (Lentinus edodes (Berk) Sing) growers

A 42‐year‐old female shiitake grower was investigated to clarify the etiology of skin lesions which developed during the planting Of shiitake hyphae into bed logs. She complained of repeated eczematous skin lesions during the planting season, from March to July, for 10 years. She handled 7,000 pieces of small conic Mucks made of beech, with shiitake hyphae attached in their surface, per day, and 300,000 pieces altogether per season. She was positive on patch testing with extracts of shiitake hyphae. In contrast, female shiitake growers with skin lesions associated with work other than planting, and without skin lesions, were negative on patch testing to she hyphae. Moderate allergenicity was observed to extracts of shiitake hyphae in a guinea pig maximization test. These findings indicated the etiology of skin lesions in shiitake growers to be allergic contact dermatitis induced by shiitake hyphae.

Immediate type of allergy in statis growers

Three statis growers complaining of immediate allergic symptoms induced by harvesting statis in a plastic greenhouse were examined to clarify their allergic conditions. Distinct positive reactions in the intradermal test and nasal and eye provocation test to the statis extracts were shown in all three cases. High score of radioallergosorbent test (RAST) IgE and remarkable RAST inhibitory effects to the statis extracts were seen in these cases. The immunologic cross-reactivity between statis and chrysanthemum was absent in the RAST inhibition in the patient labeled case C who had complained of the same allergic symptoms when handling chrysanthemums in the off-season for harvesting statis. Those results indicated that the allergic conditions of the present cases were from an immediate type of allergy mediated by a specific IgE antibody to the statis extract.

Expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde

In this study, we have investigated the expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde using semiquantitative RT-PCR. We show that formaldehyde induced the long-lasting expression of IL-4 and IFN-gamma mRNAs and the transient expression of IL-13 mRNA in mouse spleen and draining lymph nodes. The transient increases in IL-2, IL-15, IL-12p40, IL-15 and IL-18 mRNAs, but long-lasting IL-15 mRNA were only seen in the formaldehyde-exposed mouse spleen. Moreover, a weak contact hypersensitivity (CH) and the significant increases in IL-4 and IFN-gamma mRNAs were detected in the ear skin of formaldehyde-cutaneously exposed mice when rechallenged mouse ears. Furthermore, CH as measured by mouse ear swelling response was positively correlated with IL-4 and IFN-gamma mRNA levels in the challenged ears. This study thus suggests that the induction of Th1 and Th2 cytokine mRNAs, particularly IL-4 and IFN-gamma, are a common immunological feature caused by contact allergens irrespective of strong or weak contact allergens. The analysis of IL-4 and IFN-gamma mRNAs may be useful markers in establishing the novel test for predicting chemical sensitizing potentials.