PREMATURE MENOPAUSE AND OBESITY DUE TO OOCYTE LOSS IN FEMALE MICE CHRONICALLY EXPOSED TO LOW DOSE-RATE γ-RAYS.

In previous reports, the authors showed a significant overall increase in neoplasms originating from the ovaries (2007) and increased body weights (2007, 2010) in female B6C3F1 mice chronically exposed to low dose-rate γ-rays at 20 mGy/day (total doses = 8 (2007) or 6 Gy (2010)), as well as significant increases in serum leptin, total cholesterol, adipose tissue deposits and liver lipid content (2010). The present study chronicles the progression of ovarian failure in relation to obesity and dyslipidemia in female B6C3F1 mice chronically exposed to low dose-rate of γ-rays from 9 to 43 weeks of age (total dose = 4.8 Gy). We monitored changes in body weights, estrus cycles, ovarian follicle counts, serum cholesterol and serum leptin. The number of mice with irregular estrus cycles and increased body weights (with increased fat deposits) significantly increased from 30-36 weeks of age. Depletion of oocytes in ovaries from irradiated mice at 30 weeks of age (accumulated dose = 3 Gy) was also observed. Findings suggest that obesity in female B6C3F1 mice continuously irradiated with low dose-rate of γ-rays at 20 mGy/day is a consequence of premature menopause due to radiation-induced oocyte depletion.

Effects of Continuous Gamma-Ray Exposure In Utero in B6C3F1 Mice on Gestation Day 18 and at 10 Weeks of Age.

Pregnant C57BL/6JJcl mice were exposed to γ rays at low dose rate (20 mGy/day, LDR) or medium dose rate (200 and 400 mGy/day, MDR) from gestation day (GD) 0-18 to total accumulated doses of 360, 3,600 and 7,200 mGy, respectively. An additional group of pregnant mice were acutely exposed to 2 Gy at high dose rate (HDR) of 0.77 Gy/min on GD 11. In experiment 1, fetuses collected via cesarean section on GD 18 were examined for external and skeletal abnormalities. While the results of LDR exposure (20 mGy/day) did not significantly differ from the nonirradiated controls in all parameters examined, MDR (200 and 400 mGy/day) and acute HDR (2 Gy) exposure caused growth retardation and significantly increased incidence of unossified bones. Increased incidence of external abnormalities was observed only in the acute HDR group. In experiment 2, the dams were allowed to give birth and the pups were clinically monitored and weighed periodically until 10 weeks of age when they were sacrificed and subjected to pathological examination. Pups exposed at MDRs of 200 and 400 mGy/dayand at acute HDR of 0.77 Gy/min had lower bodyweights from weaning (3 weeks) to 10 weeks of age except for females exposed to 400 mGy/day MDR. None of the pups exposed to an acute 2 Gy dose on GD 11 survived to 10 weeks of age. Histopathological changes were not significantly different between the nonirradiated control and the 20 mGy/day LDR groups. However, at both MDR exposures of 200 and 400 mGy/day. gonadal (testes and ovary) hypoplasia/atrophy was observed in all the 10-week-old pups. Our results show that in utero LDR exposure to 20 mGy/day for the entire gestation period did not cause any significant effect in pups when compared to the nonirradiated controls up to 10 weeks of age. However, pups exposed in utero to MDRs showed dose-related growth retardation with delayed ossifications (400 mGy/day) and gonadal hypoplasia/atrophy. These findings suggest that increased post-implantation loss in dams exposed at MDR is due to early embryonic deaths resulting in early resorption.

Pathology of Serially Sacrificed Female B6C3F1 Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays.

We have previously reported on life span shortening as well as increased incidence rates in several neoplasms in B6C3F1 mice that were continuously exposed to 21 mGy/day of gamma rays for 400 days. To clarify whether the life shortening was due to early appearance of neoplasms (shortened latency) or increased promotion/progression, 8-week-old female specific-pathogen-free B6C3F1 mice were gamma-ray irradiated at a low dose rate of 20 mGy/day for 400 days. At 100 days postirradiation, 60-90 mice were sacrificed, and thereafter every 100 days alongside the age-matched nonirradiated controls, for 700 days. Additional groups were allowed to live out their natural life span. Pathological examination was performed on all mice to identify lesions, non-neoplastic and neoplastic, as well as to determine the cause of death. Body weights were significantly increased in irradiated mice from sacrifice days 200-500. Incidence rates for spontaneously occurring non-neoplastic lesions, such as adrenal subcapsular cell hyperplasia, fatty degeneration of the liver, atrophy and tubulostromal hyperplasia of the ovaries, were significantly increased in irradiated mice. Significantly increased incidence rates with no shortening of latency periods were observed in irradiated mice for malignant lymphomas, hepatocellular adenomas/carcinomas, bronchioloalveolar adenomas, harderian gland adenoma/adenocarcinoma. Shortened latencies with significantly increased incidence rates were observed for adrenal subcapsular cell adenomas and ovarian neoplasms (tubulostromal adenoma, granulosa cell tumors) in irradiated mice. Life span shortening in mice exposed to 20 mGy/day was mostly due to malignant lymphomas. Multiple primary neoplasms were significantly increased in mice exposed to 20 mGy/day from sacrifice days 400-700 and in the life span group. Our results confirm that continuous low-dose-rate gamma-ray irradiation of female B6C3F1 mice causes both cancer induction (shortened latency) and promotion/progression (early death), depending on the neoplasm's organ/tissue of origin.

Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells.

We developed a convenient method to methylate all CpG dinucleotides in both strands in a selected region of a plasmid, and investigated the effect of DNA methylation in the transcribed regions of reporter genes on the transient expression in HeLa cells. In a construct containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early promoter, methylation in the CAT structural gene repressed CAT activity. Methylation in the transcribed region of the Escherichia coli lacZ gene driven by the human cytomegalovirus immediate early promoter also inhibited expression of beta-galactosidase activity. These results suggest that methylation in the transcribed region as well as promoter methylation may affect transcription.

Suppressive effects of linoleic acid on neutrophil oxygen metabolism and phagocytosis.

On the basis of recent reports that the proportion of linoleic acid (C18:2Cis 9,12), a free fatty acid, is markedly decreased in acne comedones and that tetracycline is effective against acne comedones by acting directly as an antioxidant on infiltrating neutrophils, we investigated the effect of linoleic acid on several inflammatory parameters of neutrophils, including neutrophil chemotaxis, phagocytosis, and generation of reactive oxygen species (ROS). Linoleic acid significantly decreased phagocytosis and the generation of O2-, H2O2, and OH.by neutrophils, whereas it did not significantly inhibit neutrophil chemotaxis or decrease the ROS levels generated in a cell-free, xanthine-xanthine oxidase system. The present study seems to suggest that decreased levels of linoleic acid in acne comedones contribute, in part, to the worsening of acne inflammation by the failure of low levels of linoleic acid to suppress neutrophil phagocytosis and ROS generation.

Brain-to-blood active transport of beta-alanine across the blood-brain barrier.

A high-affinity antiluminal uptake system for beta-alanine was demonstrated in primary cultured bovine brain capillary endothelial cells (BCEC) for which K(t) is 66.9 microM. beta alanine uptake was energy-, sodium- and chloride ion-dependent. beta-amino acids strongly inhibited the uptake, while alpha- and gamma-amino acids had a little or no inhibitory effect. In ATP-depleted cells, the uptake was stimulated by preloading beta-alanine or taurine but not by L-leucine. These results suggest that beta-alanine is actively transported across the antiluminal membrane of BCECs that is common to beta-amino acids. The system may function for the efflux from the brain to blood.

Age-dependent alterations in mRNA level and promoter methylation of collagen alpha1(I) gene in human periodontal ligament.

In an attempt to understand the molecular mechanisms of age-dependent degenerative alteration in human periodontal tissues, we examined mRNA level and DNA methylation of collagen alpha1(I) gene. Using healthy periodontal ligament tissues from humans aged 9-76 years, we found that the collagen alpha1(I) mRNA level decreased almost linearly with age. It was observed in both Northern blot and dot blot hybridization. Examination of DNA methylation in the collagen alpha1(I) gene promoter region by its susceptibility to methylation-sensitive restriction enzyme followed by Southern blot analysis showed age-dependent increase of DNA methylation at -1705 and -80 positions located upstream of the gene. The data suggest the possible importance of alterations in collagen alpha1(I) gene expression and its DNA methylation in promoter region in age-dependent degeneration of periodontal ligament.

Molecular nature of mutations induced by a high dose of x-rays in spleen, liver, and brain of the lacZ-transgenic mouse.

DNA sequences of 103 spontaneous mutants and 102 X-ray-induced mutants of the lacZ transgene from spleen, liver, and brain of the MutaMouse were examined and compared to elucidate characteristics of radiation-induced mutations in vivo. The radiation-induced mutants were isolated from genomic DNA of each tissue collected at 3.5 days after 200 Gy of whole body irradiation. Base substitution was predominant (80% or more) in nonirradiated tissues, while deletion was prevalent (about 55%) in irradiated tissues. The other types of mutation appeared at similar frequencies in both control and irradiated tissues. The size of the deletions was smaller than 438 nucleotides, with a predominance of one basepair deletions in both control and irradiated tissues. A close look at the nucleotides at the deletion endpoints revealed that many of the radiation-induced deletions did not have repeated sequences at the break point termini, whereas all deletions found in unirradiated tissues showed one or more bases of repeated sequences at the termini. Further, eight complex-type deletion mutations were found only in irradiated tissues. Comparison among the three types irradiated tissue did not reveal any tissue-specificity. The data indicate that the molecular nature of mutations induced in tissues with ionizing radiation is different from that of spontaneous mutations.

Linear unilateral basal cell nevus with comedo-like lesions.

A 20-year-old woman had linear unilateral basal cell nevus with comedo-like lesions. Numerous papules were distributed on the left side of the upper aspect of the trunk in a zosteriform fashion. The indivisual lesions were hemispherical, pearly, and waxy tumors, some of which had central plugs. Histologic examination revealed polymorphous patterns that suggested that the tumor cells differentiated toward the apocrine glands, hair structures, and sebaceous glands. Ultrastructurally, the tumor strands were composed of fairly mature basal cells with well-developed tonofilaments and desmosomes.

True histiocytic lymphoma.

An 86-year-old man had true histiocytic lymphoma of the skin. Diagnosis was established by the following findings: (1) typical histological features, (2) lack of receptors on tumor cells for immunological surface markers, and (3) active phagocytic function. Electron microscopic studies indicated cytoplasmic multivesicular bodies in the tumor cells. Multivesicular bodies have been reported in histiocytic proliferative disorders and may be of diagnostic value in cases of true histiocytic lymphoma.

Neutrophil-generated active oxygens in linear IgA bullous dermatosis.

To examine the possible correlation between tissue injury and neutrophil-produced active oxygens (AOs) in patients with linear IgA bullous dermatosis (BD), we studied the capacity of neutrophils from six patients with BD to generate AOs. Cultured endothelial cells from human umbilical-cord vein were also incubated with the patients' neutrophils to assess AO-induced tissue injury. The AO production by patients' neutrophils was significantly elevated. The patients' neutrophils, as well as those from healthy controls preincubated with patients' serum, produced significantly increased levels of cytotoxic response on coincubation with chromium 51-labeled human endothelial cells. These results suggest that the tissue damage observed in BD may be partially due to both excessive production of AOs by neutrophils and a serum factor present in the patients, and further postulate the similar pathogenic process in dermatitis herpetiformis.

More efficient excision repair of pyrimidine dimers in the specific DNA sequence than in the genome overall in goldfish cells.

Excision repair of pyrimidine dimers induced by 254 nm UV was examined in the genome overall and in a specific sequence containing a transfected gene for hygromycin B resistance, in RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. More than 40% of dimers were removed from the specific sequence, while about 20% were removed from the genome overall, within 24 h after exposure to UV (2.5-7.5 J/m2).

The effect of aging on cutaneous lipid peroxide levels and superoxide dismutase activity in guinea pigs and patients with burns.

Cutaneous lipid peroxide levels and superoxide dismutase (SOD) activity in non-aged and aged guinea pigs were measured between 15 min and 7 days after experimental infliction of burns. Skin burns on non-aged and aged patients were also subjected to these assays. In non-aged guinea pig skin burns, lipid peroxide levels increased from 24 hr to the fourth day after the burn infliction, while SOD activity did not increase but showed a slight decrease 12 hr and 24 hr post-burn. On the other hand, while the aged group showed a more increase in skin lipid peroxide levels compared to that seen in non-aged mice, skin SOD activity began to decrease from 30 min post-burn, the maximum decrease being reached on the second day. The activity did not return to normal by the 7th day. In non-aged patients skin burns showed increases in both lipid peroxide levels and SOD activity, while in aged patients, though they showed a marked increase in lipid peroxide levels, SOD activity remained unchanged. The present study indicated that, although in our recent study, skin SOD activity of healthy elderly people was found to be comparable to that in non-aged individuals, the capacity for induction of SOD activity under oxygen stress differed with age in both guinea pig and human burn sufferers. Furthermore, this induction capacity seemed to vary from species to species.

Radiation-induced mutations in the spleen and brain of lacZ transgenic mice.

PURPOSE: To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. MATERIALS AND METHODS: Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. RESULTS: X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. CONCLUSIONS: Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.

Ultrastructural studies of allergic contact dermatitis in man. Epidermal cell changes at 3, 6, and 12 h after application of DNCB.

Early epidermal lesions of allergic contact dermatitis were examined by electron microscopy. Normal human volunteers were sensitized to DNCB, and contact reactions were elicited sequentially. Epidermal cell changes at 3 h included: the occurrence of small vacuoles with or without membrane, focal dilatation of intercellular spaces, and the alteration of tonofilaments into short, aggregated bundles. Non-sensitized skin to which DNCB was applied also showed small vacuoles with or without membrane. Monocytes infiltrated into the intercellular spaces which were not dilated, and the neighboring tonofilaments of keratinocytes remained normal.

Ultrastructural studies of epidermis in acute radiation dermatitis. Basal lamina thickening and coated vesicles.

Fine structural changes of keratinocytes by X-ray were examined in normal skin area around a lesion of Bowen's disease. The area was exposed to about 6000 r over 28 days. The findings were: a decreased number of desmosomes and microvilli, formation of cytoplasmic vaculoes with or without membrane, perinuclear aggregation of tonofibrils, intracytoplasmic desmosomes and gap junction, cytoplasmic occurrence of dense bodies, lipid droplet and glycogen particles, changes in mitochondria, endoplasmic reticulum and Golgi complex, and deep invagination of the nuclear membrane. Besides these, conspicuous in the present study were, changes in the basal lamina which consisted of multiplication, thickening and occasional detachment from basal cells, and frequent occurrence of coated vesicles along with the basal lamina alteration. Possible exocytotic nature of coated vesicles in X-ray irradiated keratinocytes is discussed.

Experimental burn-induced changes in lipid peroxide levels, and activity of superoxide dismutase and glutathione peroxidase in skin lesions, serum, and liver of mice.

The lipid peroxide levels and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in skin lesions, liver, and serum of mice were measured from the 15th min to 6th day after burns were inflicted on their skin. The lipid peroxide levels in the skin lesions were increased at the 24th h and on the 3rd day after the burns were inflicted. However, the SOD activity did not increase but was significantly decreased from the 30th min to 24th h. GSH-Px activity was almost undetectable in both skin and serum. The lipid peroxide levels in the serum were significantly increased and SOD activity slightly increased at the 4th h. The lipid peroxide levels in the liver were elevated at the 4th h and on the 3rd day although a slight decrease was observed at the 8th h. SOD and GSH-Px activities in the liver increased from the 4th to 24th h. The present study illustrates the changes in both burn-induced lipid peroxides in each organ and the induction mechanisms of the activities of oxygen radical scavenging enzyme, SOD, and lipid peroxide processing enzyme, GSH-Px, which responded to oxygen stress.

Ultrastructural studies of allergic contact dermatitis in man. Infiltrating cells at the earliest phase of spongiotic bulla formation.

The kind and fine structure of mononuclear cells appearing in the epidermis at about 6 h of allergic contact dermatitis were examined by electron microscopy. They were monocytes and lymphocytes, the number being about equal. The ultrastructure of monocytes was that described for normal ones in blood, and apparently actively moving, streching the intercellular connections of the keratinocytes. Lymphocytes displayed a round or oval nucleus with some electron-dense cytoplasm which contained ribosomes and polysomes but only occasional mitochondria and Golgi complexes.

Ultrastructural studies of spontaneously regressing plane warts. Langerhans cells show marked activation.

Ultrastructural changes in Langerhans cells during spontaneous involution of plane warts were examined. In areas with activated macrophages and epidermal cell interaction, Langerhans cells showed signs of enhanced cellular activity with an increased number of Langerhans cell granules, as in contact dermatitis. The fine structure of Langerhans cell granules, however, was unusual in that the vesicular portion was surrounded by a membrane, and this portion frequently occurred independently taking the shape of a loop.

Ultrastructural studies of spontaneously regressing plane warts. Macrophages attack verruca-epidermal cells.

Plane warts were examined at the earliest phase of spontaneous involution using an electron microscopy. Macrophages outnumbered lymphocytes in invading the epidermis. Lymphocytes exhibited pseudopods on the surface which occasionally reached into the epidermal cells. The cell membranes of both macrophages and epidermal cells frequently disappeared on the contacting surface and on occasion part of the cytoplasm and even the nucleus of macrophages invaded the epidermal cells. The macrophages showed an independent partial loss of their cell membranes with a subsequent outpour of their contents. This ultrastructural feature indicates that in spontaneously regressing plane warts the depressed membrane stability of macrophages as observed in BCG and other-induced tumor cell destruction also occurs in the presence of lymphocytes. Therefore, the spontaneous involution may represent a rejection phenomenon.