Magnesium augments immunosuppressive effects of a corticosteroid in obese mice with airway inflammation.

BACKGROUND: Magnesium deficiency common in obesity is known to promote chronic low-grade inflammation and aggravate asthma symptoms; however, the effects of magnesium supplementation in obese asthmatic patients have not been investigated. OBJECTIVE: To examine the effects of magnesium co-administration with dexamethasone on airway inflammation in obese mice. METHODS: Female C57BL/6 mice were fed a high-fat diet, sensitized with ovalbumin (OVA) to induce allergic reactions, challenged with aerosolized OVA, and administered dexamethasone (3 mg/kg) with or without magnesium. Bronchial inflammation was analyzed based on the presence of inflammatory cells and cytokines in bronchoalveolar lavage fluid, total and OVA-specific IgE in serum, goblet cells ratios, bronchial wall thickness, and expression of α-smooth muscle actin. RESULTS: In obese mice, co-administration of magnesium and dexamethasone decreased IL-13 in bronchoalveolar lavage fluid and total and OVA-specific IgE in serum, and reduced α-smooth muscle actin-positive areas in the bronchi compared with mice treated with dexamethasone alone. However, no differences were observed in dexamethasone-treated normal-weight mice depending on magnesium supplementation. CONCLUSION: These results suggest that magnesium increases immunosuppressive effects of dexamethasone in airway inflammation aggravated by obesity, suggesting that magnesium supplementation may have a potential in alleviating asthma symptoms in obese patients with reduced responses to corticosteroids.

Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on susceptibility to neonatal asthma.

Di(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer and is widely dispersed in the environment. In this study, we investigated the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using a murine model of asthma induced by ovalbumin (OVA). Pregnant BALB/c mice received DEHP from gestation day 13 to lactation day 21. Their offspring were sensitized on postnatal days (PNDs) 9 and 15 by intraperitoneal injection of 0.5µg OVA with 200µg aluminum hydroxide. On PNDs 22, 23 and 24, live pups received an airway challenge of OVA for 30min. Offspring from pregnant mice that received DEHP showed reductions in inflammatory cell count, interleukin (IL)-4, IL-13, and eotaxin in their bronchoalveolar lavage fluid and in total immunoglobulin E and OVA-specific IgE in their plasma compared with offspring from pregnant mice that did not receive DEHP treatment. These results were consistent with histological analysis and immunoblotting. Maternal exposure to DEHP reduces airway inflammation and mucus production in offspring, with a decrease in inducible nitric oxide synthase (iNOS) in the lung tissue. This study suggests that maternal exposure to DEHP during pregnancy reduces asthmatic responses induced by OVA challenge in offspring. These effects were considered to be closely related to the suppression of Th2 immune responses and iNOS expression.

Promoting potential of a Jamaica quassia extract in a rat medium-term hepatocarcinogenesis bioassay.

Jamaica quassia extract (JQE), a natural bittering agent, was investigated for hepatocarcinogenesis-promoting potential using a medium-term liver bioassay system. F344 male rats were given a single intraperitoneal injection of diethylnitrosamine (200mg/kg body weight) and then starting 2 weeks later, received JQE in the diet at concentrations of 500, 5000 or 30,000 ppm for 6 weeks. Animals for tumor promotion (+) and (-) controls were fed 500 ppm sodium phenobarbital (PB) and basal diet, respectively during the promotion phase in this model. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. As with the PB-promoted case, both numbers and areas of glutathione S-transferase placental form-positive liver cell foci were significantly increased by JQE at 30,000 ppm, with non-significant increases evident at 5000 ppm. The results thus indicate that JQE at high dose has promoting potential for rat hepatocarcinogenesis.

Lack of preventive effects of dietary fibers or chlorophyllin against acrylamide toxicity in rats.

Dietary fibers and chlorophyllin have shown to exert anti-carcinogenic effects against co-administered carcinogens. To test the possibility of chemoprevention by such dietary supplements on subacutely induced acrylamide (ACR) toxicity, Sprague-Dawley male rats were administered 2.5% sodium alginate, 5% glucomannan, 5% digestion resistant maltodextrin, 2.5% chitin or 1% chlorophyllin in the diet, and starting one week later, co-administered 0.02% ACR in the drinking water for 4 weeks. For comparison, untreated control animals given basal diet and tap water were also included. Neurotoxicity was examined with reference to gait abnormalities and by quantitative assessment of histopathological changes in the sciatic and trigeminal nerves, as well as aberrant dot-like immunoreactivity for synaptophysin in the cerebellar molecular layer. Testicular toxicity was assessed by quantitation of seminiferous tubules with exfoliation of germ cells into the lumen and cell debris in the ducts of the epididymides. Development of testicular toxicity as well as neurotoxicity was evident with ACR-treatment, but was not suppressed by dietary addition of fibers or chlorophyllin, suggesting no apparent beneficial influence of these dietary supplements on experimentally induced subacute ACR toxicity.

Regional differences in vulnerability of the cerebellar foliations of rats exposed to neonatal X-irradiation.

The purpose of the present study was to elucidate regional differences in the vulnerability of cerebellar foliations of rats exposed to X-irradiation. Effects of X-irradiation on foliations were examined with respect to histological changes in Purkinje cells and Bergmann glial fibers by calbindin-D28k (CB) and glial fibrillary acidic protein (GFAP) immunohistochemistry, respectively. Wistar rats were exposed to X-irradiation (1.5 Gy) on postnatal day (PND) 1. At 3 weeks of age, the cerebellum was examined. The cerebella of rats exposed to X-irradiation showed smaller and abnormal foliations compared with controls. Fewer cerebellar foliations due to fusion with neighboring folia were observed in folia I-III and VIa-VII. Moreover, the extent of such abnormalities was more severe in the latter folia. CB-immunoreactive (IR) Purkinje cells exhibited thin, short, disoriented dendrites that had invaded the granular layer or white matter. On the other hand, GFAP-IR Bergmann glial fibers had not extended their processes into the molecular layer perpendicular to the pial surface, and they appeared thin and disoriented. Accordingly, the above cerebellar abnormalities were more severe in folia I-III, VIa-VII and X than in other regions. In contrast to the histological alterations in these folia, there were no apparent qualitative differences in folia IV-V between X-irradiated and controls. These findings indicate regional difference in the vulnerability of cerebellar folia to X-irradiation. Such differences might be attributed to the cerebellar neurogenetic gradient.

Oligodendrocyte myelin glycoprotein (OMgp) in rat hippocampus is depleted by chronic ethanol consumption.

The hippocampal formation has been shown to be particularly vulnerable to the neurotoxic effects of chronic ethanol consumption. It was hypothesized that this damage was due to the disruption of the expression of neurotrophic factors and certain other proteins within the hippocampus. By using real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques, this study aimed to determine whether chronic ethanol consumption could alter the mRNA expression level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and oligodendrocyte myelin glycoprotein (OMgp) in the hippocampus. Wistar male rats received an unrestricted access to a liquid diet containing 5% (v/v) ethanol as the sole source of fluid from 10 to 29 weeks of age. Control rats had unlimited access to a liquid diet containing an isocaloric amount of sucrose. We found that chronic ethanol consumption did not cause significant changes in the levels of mRNA for BDNF and GDNF. However, OMgp mRNA showed a significant deficit in ethanol-treated animals. It is suggested that this deficit may be related to the demyelination that is commonly observed in human alcoholics and that this may contribute to the functional and cognitive deficits.

Subchronic toxicity study of water pepper extract in F344 rats.

A subchronic toxicity study of water pepper extract (WPE) from Polygonum hydropiper L. was conducted in groups of 10 male and 10 female F344 rats fed powdered diets containing 0, 62.5, 250, 1000 or 4000 ppm concentrations for 13 weeks. Suppression of body weight gain due to decreased food consumption was observed in both sexes at 4000 ppm, and at autopsy, increase of relative weights was observed for the brain, liver, spleen, kidneys, and testes in these animals, suggestive of the reflection of the reduced body weights. At this dose, slight increases of blood urea nitrogen in both sexes and serum alanine aminotransferase, Na and Cl in females, were observed, suggestive of weak hepatic and renal toxicity, at least in females. The same females also exhibited slight decrease of red blood cells and haematocrit, slight increase of mean corpuscular volume and mean corpuscular haemoglobin, and minimal increase of splenic haemosiderin deposition, providing evidence of slight haemolytic anemia. On the other hand, enhanced accumulation of mast cells was observed in the mesenteric lymph nodes at 4000 ppm in males and 1000 and 4000 ppm in females. Considering the anti-anaphylactic properties of polygodial, a major constituent of WPE, the mast cell accumulation was concluded to be an adaptive change in response to the subchronic oral administration of WPE. Based on the present toxicity data, 1000 ppm was determined to be the no-observed-adverse-effect level, translating into 57.4 and 62.9 mg/kg/day for male and female rats, respectively.

Age-related changes in growth hormone-immunoreactive cells in the anterior pituitary gland of Jcl: Wistar-TgN (ARGHGEN) 1Nts rats (Mini rats).

Rats of the Jcl: Wistar-TgN (ARGHGEN) 1Nts strain (Mini rats) are transgenic animals carrying an antisense RNA transgene for rat growth hormone (GH); they show poor somatic growth and a low blood GH level compared to age-matched wild-type Wistar (non-Mini) rats. The purpose of the present study was to investigate age-related changes in growth hormone-immunoreactive (GH-IR) cells in the anterior pituitary gland (AP) of Mini rats at four, six, and eight weeks of age. The body weight and size of the GH-IR cells of Mini rats was significantly lower than that of non-Mini rats at six and eight weeks of age; however, this difference was not observed at four weeks of age. The AP volume and the number of GH-IR cells in Mini rats were significantly smaller than those of the age-matched non-Mini rats at the three ages. These results suggest that the abnormal development of GH-IR cells in the AP induced by the GH antisense RNA transgene is responsible for the poor somatic growth and the low blood GH levels in Mini rats.

Effects of chronic ethanol administration on the expression levels of neurotrophic factors in the rat hippocampus.

Chronic ethanol consumption has adverse effects on the central nervous system. Hippocampus is one of the target sites of ethanol neurotoxicity. Hippocampal damage is known to result in impairment of learning and memory. This study was aimed to determine whether chronic ethanol consumption could alter the expression levels of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) mRNAs in the hippocampus. Male Wistar rats were given unrestricted access to a liquid diet containing 5% (v/v) ethanol as the sole fluid source for 19 weeks beginning at 10 weeks of age. The expression levels of BDNF and GDNF mRNAs in the hippocampus were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. The present study revealed that chronic ethanol consumption did not result in significant changes in the expression levels of BDNF and GDNF mRNAs. Our present results showed no significant alteration in the expression of these neurotrophic factors; these results will lead to further studies to examine the possible alterations in the gene expression of various neurotrophins that are related to hippocampal functions including learning and memory.

Limited tumor-initiating activity of phenylethyl isothiocyanate by promotion with sodium L-ascorbate in a rat two-stage urinary bladder carcinogenesis model.

Initiation activity of phenylethyl isothiocyanate (PEITC) was examined in a two-stage urinary bladder carcinogenesis model. Male 6-week-old Fischer 344 rats were fed diet containing 0.1% PEITC for 12 or 24 weeks, with or without subsequent administration of 5% sodium l-ascorbate (Na-AsA) in diet until week 48, or for the entire experimental period. After 12 weeks of PEITC-treatment, both simple hyperplasia and papillary or nodular (PN) hyperplasia had developed in all animals, but the majority of these lesions had disappeared at week 48, irrespective of the Na-AsA-treatment. The same lesions after 24 weeks of PEITC-treatment had progressed to dysplasia and carcinoma, in a small number of cases by week 48 (6% in incidence for each lesion), but enhancement by the Na-AsA-treatment was evident only with simple hyperplasias (from 56 to 100% in incidence) and PN hyperplasias (from 19 to 56%). The results suggest a limited initiation activity of PEITC with induction of irreversible lesions by 24 weeks of exposure.

Application of the physical disector to the central nervous system: estimation of the total number of neurons in subdivisions of the rat hippocampus.

Stereology is a group of mathematical and statistical methods that allows the extrapolation of three-dimensional structural information from two-dimensional sections (or slices). This allows researchers to derive important quantitative structural information, such as the volume, surface area or numbers of particular particles (e.g. cells) within defined regional boundaries. The need for such quantitative information in biology is of particular importance when evaluating the influence of various experimental treatments on specific organs, tissues and cells in the body. Knowledge of such changes has given important insights into the neural substrates that may be responsible for the functional and behavioral consequences of a disparate range of experimental treatments. Here, we describe some of these methods as applied to quantifying the total numbers of cells in defined regions of the hippocampal formation. The methods used for this evaluation were, first, the Cavalieri principle, which was used to determine the volumes of the various subdivisions of the rat hippocampus, and, second, the 'physical disector' method, which was used to estimate the numerical density of neurons within each subdivision. Once these values were derived, it was but a simple task to multiply them together to obtain estimates for the total numbers of cells in the given hippocampal region. We found that 16-and 30-day-old normal male rats had 176 800 and 152 700 pyramidal cells in the CA1 region, respectively. This decrease in the neuronal number was statistically significant. However, in the CA2 + CA3 region, there were approximately 169 300 and 149 600 pyramidal cells in 16- and 30-day-old normal male rats, respectively, which was not significantly different. In the dentate gyrus, there were approximately 36 700 neurons in the hilus region and 483 000 granule cells in the granule cell layer, irrespective of the age of the rats. There were no significant differences between these estimates of hilus neurons and granule cells.

Down-regulation of GAT-1 mRNA expression in the microdissected hypothalamic medial preoptic area of rat offspring exposed maternally to ethinylestradiol.

Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.

Effects of monocular enucleation on calbindin-D 28k and c-Fos expression in the lateral geniculate nucleus in rats.

The present study was undertaken to evaluate the effects of monocular enucleation on the calbindin-D 28k (CB) and c-Fos immunoreactive (IR) neurons in the lateral geniculate nucleus (LGN) complex of adult rats. The enucleation resulted in neuronal degeneration and decrease of neurons in the LGN complex. Our study demonstrated a decrease of CB-IR neuronal density on the contralateral side of the ventral (vLGN) and dorsal LGN (dLGN) until 12 weeks post-enucleation (PE). On the ipsilateral side, CB-IR neuronal density in the dLGN and vLGN showed significant and continuous decrease until 48 and 12 weeks PE, respectively. In an additional experiment, c-Fos-IR neurons were increased at 2 days PE in the vLGN with ipsilateral predominance. At 7 days, c-Fos-IR neurons on the ipsilateral vLGN were still higher than those of pre-enucleated rats. The present results suggest that monocular enucleation affects the expression of the CB and c-Fos in the LGN complex. It is indicated that these may play an important role in the neuronal degeneration and neuroplasticity of the subvisual system in rats.

Microdissected region-specific gene expression analysis with methacarn-fixed, paraffin-embedded tissues by real-time RT-PCR.

We have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients >0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, >/=200 cells were necessary for accurate measurement in 10-microm-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.

Cdk5/p35 expression in the mouse ovary.

Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.

Diverse developmental toxicity of di-n-butyl phthalate in both sexes of rat offspring after maternal exposure during the period from late gestation through lactation.

To evaluate developmental toxicity of di-n-butyl phthalate (DBP) with exposure during the period from late gestation to following lactation, maternal rats were given DBP at dietary concentrations of 0, 20, 200, 2000 and 10,000 ppm from gestational day 15 to postnatal day (PND) 21. At 10,000 ppm, male offspring showed a decreased neonatal anogenital distance and retention of nipples (PND 14), while females showed a slight non-significant delay in the onset of puberty. At PND 21, reduction of testicular spermatocyte development was evident from 20 ppm, as well as mammary gland changes at low incidence in both sexes. At this time point, population changes of pituitary hormone-immunoreactive cells were observed at 10,000 ppm with a similar pattern of increase in the percentages of luteinizing hormone (LH)-positive and decrease in follicle-stimulating hormone (FSH) and prolactin producing cells in both sexes, effects also being evident on FSH from 200 ppm and LH from 2000 ppm in females. During postnatal week (PNW) 8-11, marginal increase of the number of cases with extended diestrus was found at 10,000 ppm. At adult stage necropsy, testicular lesions appeared to be very faint in most cases, but degeneration and atrophy of mammary gland alveoli were observed in males from 20 ppm. Although without clear monotonic dose-dependence, relative pituitary weights were increased with the intermediate doses in males at PNW 11. In females, relative pituitary weights were decreased after 10,000 ppm at PNW 11, and from 200 ppm at PNW 20. The proportion of FSH-positive cells in the pituitaries at PNW 11 was increased in both sexes at 10,000 ppm. Thus, developmental exposure to DBP affected female sexual development involving pituitary function, while in males testicular toxicity was mostly reversible but mammary gland toxicity was persistent at a dose level as low as 20 ppm.

Gene expression profile in the livers of rats orally administered ethinylestradiol for 28 days using a microarray technique.

To identify genes showing responses to estrogen exposure in the livers of animals in a repeated oral dose toxicity study, dose-dependent gene expression profiles were analyzed using high-density oligonucleotide microarrays in Sprague-Dawley rats of both sexes administered ethinylestradiol (EE) for 28 days at concentrations of 0, 0.01, 0.1, and 1.0 ppm in the diet. Among 3776 genes examined, examples showing increased expression on EE-treatment were detected predominantly in females. Genes showing dose-dependent up-regulation with greater than five-fold change at 1.0 ppm from the control levels were found to, respectively, number 4 in males, and 24 in females. Most of the latter exhibited relatively high basal expression as well as low variability, and many exhibited clear dose-dependence. Genes showing dose-dependent down-regulation were rather few, and many of those affected exhibited relatively low expression levels with large variation between animals, like genes showing dose-unrelated expression patterns in both sexes or dose-dependent up-regulation in males. Considering that detection of changes in endocrine-linked organs and estrous cyclicity is only possible at the high dose of 1.0 ppm, up-regulation of genes dose-dependently in females provides a sensitive tool to detect estrogenic effects in the rat liver in the framework of the 28-day toxicity study.

Lack of modifying effects of genistein on disruption of the reproductive system by perinatal dietary exposure to ethinylestradiol in rats.

We previously found that effects of perinatal dietary exposure to ethinylestradiol (EE) on the rat reproductive system differ depending on the diet used, with a more pronounced estrogenic impact with a regular diet that includes soy-derived proteins than with a soy-free (SF) diet. The present study was performed to examine whether genistein (GEN), a soy-derived major phytoestrogen, acts synergistically with EE. Maternal rats were fed SF diet without chemical (control) or containing 0.5-ppm EE, 0.5-ppm EE + 100-ppm GEN, 0.5-ppm EE + 1250-ppm GEN, or 1250-ppm GEN, from gestational day 15 to postnatal day (PND) 11. EE reduced serum testosterone in males at PND 3, and affected the onset of puberty of both sexes and estrous cyclicity and reproductive system in females, irrespective of co-administration of GEN. GEN alone also affected estrous cyclicity and the reproductive system in females. However, no combination effects of GEN with EE were evident, suggesting no synergism between the two.

Subchronic toxicity study of dietary N-acetylglucosamine in F344 rats.

A subchronic toxicity study of N-acetylglucosamine (GlcNAc), a monomeric form of chitin, was conducted in groups of 10 male and 10 female F344 rats fed pelleted diets containing 0, 0.625, 1.25, 2.5 or 5% concentrations for 13 weeks. All animals survived until the end of the experiment. Slight, non-significant increase in body weights was observed in males receiving 0.625, 1.25 or 2.5% from week 4 until the end of the experiment, when significant elevation was found for the males receiving 0.625, 1.25 or 2.5% at the terminal sacrifice to result in decreased relative weights of many organs in these cases. However, there were no obvious indications of toxicity in any group receiving GlcNAc in terms of clinical signs, food intake, hematology, serum biochemistry, and histopathological findings. Thus, it was concluded that orally administered GlcNAc exerts no obvious toxicity to F344 rats at concentrations up to 5% in the diet for 13 weeks. Based on the present toxicity data, > or =5% was determined to be a no-observed-adverse-effect level, translated into 2476 and 2834 mg/kg/day for male and female rats, respectively.

Allergenicity of hen's egg ovomucoid gamma irradiated and heated under different pH conditions.

This study was conducted to evaluate the effect of a treatment combining gamma radiation and heating on the allergenic properties of hen's egg ovomucoid (OM) under basic pH conditions. OM solutions of 2.0 mg/ml with pHs of 7.0, 9.0, and 10.0 were gamma irradiated at 10 kGy, heated at 100 degrees C for 15 min, or both. Half of the treated pH 10.0 sample solution was restored to pH 7.4 by dialysis. OM solutions were tested by a competitive direct enzyme-linked immunosorbent assay formatted with immunoglobulin E from egg-hypersensitive patients. An equation was obtained for quantifying intact OM from the standard curve, and the detected concentration of intact OM was calculated. The concentration of intact OM decreased with irradiation or heating, and the rate of the decrease was higher for a basic pH condition than for the physiological condition. The combination of irradiation and heating was very effective in reducing the amount of intact OM regardless of the pH condition. After treatment, the restoration of the pH to 7.4 did not affect the concentration of OM. The results of this study indicate that a combination of irradiation and heating might be an effective method for reducing egg hypersensitivity resulting from OM.