Mikako Tsukada

Evidence on n -acetyltransferase Allele-associated Metabolism of Hydrazine in Japanese Workers

Hydrazine (N2H4), which has been categorized as a weak carcinogen, is a chemical with the one of the largest production rates in Japan. We have investigated the effects of acetylation phenotypes on the metabolism of hydrazine. Genotypes of N-acetyl transferases, NAT2*, were determined using polymerase chain reaction for 297 male workers. Biological and exposure monitoring were also conducted. The rapid and intermediate acetylators accounted for 45% each, and the slow acetylators accounted for 10%. Biological half-lives were significantly different among the three acetylation phenotypes (analysis of variance, P < 0.05): 3.94+/-1.70 hours for slow acetylators, 2.25+/-0.37 hours for intermediate acetylators, and 1.86+/-0.67 hours for rapid acetylators. Among Japanese, rapid and intermediate acetylators are the major phenotypes, which is in sharp contrast with those among Caucasians. We conclude that biological monitoring should take genetic factors, which may vary dramatically among different populations, into account.

Low blood glucose levels and small islets of Langerhans in the pancreas of calorie-restricted mice

In the present study we investigated the influence of long-term caloric restriction on blood glucose levels and the volume of the islets of Langerhans in the pancreases of mice. Thirty female F1 mice (SHN female × C3H male) were divided into two equal groups and raised for 11 months on either a slightly restricted “control” diet (95 Kcal per week) or a more severely restricted diet (50 Kcal per week), starting at time of weaning (4 weeks of age). These diets, designated as “isonutrient” (1), were designed so that both groups of animals received approximately equal amounts of minerals, proteins, fats and vitamins, but with carbohydrates adjusted to provide the desired calorie differences. Calorie-restricted animals (CR mice) were fed 3 days a week (Monday, Wednesday and Friday) with the restricted diet at 9:00 a.m., while control animals (C mice) were fed the control diets at 9:00 a.m. every day from Monday to Friday. On Sunday any leftover food was removed from the cages at 9:00 a.m. and both CR and C animals were allowed to fast. Fasting blood glucose levels were determined in the blood collected between 9:00 and 10:00 a.m. in 12-month-old mice from both groups on Saturday and on Monday 8 days later, at which time the mice were sacrificed for histological examination. The Saturday morning blood glucose levels (in mg/100 ml of blood) were lower in CR mice than in C mice (112±12 in CR mice and 145±16 in the C mice: p<0.01). A similar trend was noted in the Monday morning samples: 80±19 in the CR and 108±28 in the C mice. In agreement with the blood glucose trends, histological examination of the pancreases demonstrated that the volume of the islets of Langerhans were significantly smaller in CR mice (Geometric mean 76646 μ3/islet, Geometric Standard deviation 2.10) than in C mice (Geometric mean 235578 μ m3/islet, Geometric Standard deviation 3.81)(p<0.01). We conclude that caloric restriction decreases not only blood glucose levels, but reduces volumes of the islets of Langerhans.

Caloric restriction perturbs the pituitary-ovarian axis and inhibits mouse mammary tumor virus production in a high-spontaneous-mammary-tumor-incidence mouse strain (C3H/SHN)

Dietary restriction (DR) retards aging and extends maximum life span. It is also known to decrease the incidence of hormone-dependent tumors. In the present investigation, we focussed primarily on the influence of DR on the pituitary-ovarian axis, and subsequently on gene expression of the mouse mammary tumor virus. F1 females from the mating of SHN female and C3H male mice were used in this study, since these hybrids display a very high incidence of mouse mammary tumors. The mice weaned at 3 weeks were raised on either a calorically-restricted diet (DR: 50 kcal/week; N = 5) or on a control diet (C: 95 kcal/week N = 5) for 5 weeks. Three C57BL/6J Jcl ad libitum-fed female mice, 8 weeks of age, were used as reference animals since this strain has a very low incidence of mammary tumors. The mean cellular contents of prolactin (PRL) and growth hormone (GH) in the pituitary, as determined by immunohistochemistry, were found to be reduced in mice raised on the DR diet. The decrease in the mean cellular content of PRL (50% of the mean control value) was accompanied by a decrease in the number of lactotrophs (17% of the mean value of control diet mice). However, the decrease in cellular content of GH (53% of the mean control value) was not accompanied by a decrease in the number of somatotrophs (no. of somatotrophs in DR = 103% of mean control value). Histologically, ovaries from DR mice showed many growing and atretic follicles, with few corpora lutea. In contrast, both control-diet and reference (C57BL/6J Jcl) mice showed two or three corpora lutea per ovarian section. In accordance with this finding, DR mice had not established stable estrus cycles by 8 weeks of age, in contrast to both control-diet and reference mice. Since caloric restriction has been shown to decrease mammary tumor virus (MMTV) gene expression, MMTV production was investigated by electron microscopy to confirm the validity of our experimental conditions. In DR or reference C57BL/6J Jcl mice, MMTV particles were rarely found in the mammary gland samples, but were always found in samples from control mice. The development of mammary glands, as indicated by the number of villi or the development of the rough endoplasmic reticulum, was delayed in DR mice. Thus, it was concluded that caloric restriction decreases the number of lactrotrophs, inhibits ovulation and delays mammary gland development. This immature status is considered to be due to perturbations in the pituitary-ovarian axis by caloric restriction.

Increase in housing temperature can alleviate decreases in white blood cell counts after energy restriction in C57BL/6 female mice

White blood cell (WBC) counts decrease after energy restriction (ER) in both rodents and humans. ER reduces cellular proliferation rates in various organs of rodents. Increases in housing temperatures can cancel this inhibitory effect of ER. We investigated whether a high housing temperature can alleviate decreases in WBC counts after ER, since peripheral WBC counts at basal levels are considered to represent cellular proliferation rates in the bone marrow. C57BL/6 female mice were allocated to three groups: the control (397 kJ/week), the ER (176 kJ/week) and the ER housed-in-incubator group (ERI: 134 kJ/week). The former two groups were housed at 21-23 degrees C and the third group was housed at 30 degrees C. At 4 and 13 months of age, WBC counts were significantly lower in ER or ERI mice than in control mice. WBC counts, however, were significantly higher in ERI mice (194%) than in ER mice at 13 months of age. A high housing temperature was proved to antagonize the effect of ER on WBC counts, as it antagonizes suppressive effects of ER on cellular proliferation rates in various organs.

Specific inhibition of pituitary prolactin production by energy restriction in C3H/SHN female mice

Pituitaries were excised from control (C; 95 kcal/week) or energy restricted (ER; 48 kcal/week) female mice of 2, 3, 7, and 18 months of age. The total RNA and relative actin mRNA amounts in the pituitary were significantly greater in C than in ER mice both at 7 and 18 months. Prolactin (PRL) mRNA, standardized with actin mRNA, was significantly less in ER mice of 7 (50%) and 18 (51%) months of age than in age-matched controls, suggestive of specific inhibition of PRL mRNA transcription. Pituitary RNA and actin mRNA increased from 7 to 18 months in C mice but not in ER mice. Similarly, mean pituitary volumes increased between 2 and 18 months in C mice but not in ER mice. PRL mRNA, standardized with actin mRNA, did not change in either C or ER mice 7-18 months of age. All examined C mice of 3, 7, and 18 months of age had estrous cycles but none of the ER mice of the same ages. After 1 month of ER, the pituitary volumes and serum insulin concentrations in 2-month-old female mice were reduced. Thus net reduction of PRL mRNA per pituitary by ER is attributable to decreases in pituitary size and specific inhibition of PRL production, both of which may be due to low estrogen and insulin levels.

Retroviral gene expression as a possible biomarker of aging

SummaryWe examined effects of aging on endogenous retrovirus gene expression of mouse lymphocytes with a hypothesis that it may be a useful biomarker of aging. Mice have endogenous murine leukemia viruses (MuLVs) in their chromosomes. We detected the gene expression of long terminal repeats (LTRs) of MuLVs. Brains, livers and spleens were taken from young (3 months old) and old (27 months old) male C57BL/6 mice. In addition to these control (C) mice, we also determined gene expression in dietary restricted (DR) mice, in which rates of aging are known to be slowed. RNA was extracted from the tissues and converted into cDNA. The MuLV-LTR portion of cDNA was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by agarose gel electrophoresis. Gene expressions of young mice were found to be tissue-specific. Expressed LTRs from brains, livers and spleens were that of 370 base-pairs (bp), those of 370 and 620 bp, and those of 370, 400 and 620 bp, respectively. Old mice of C group, however, decreased tissue specificity: expressed LTRs became those of 370–400 by in any tissues. In contrast the tissue specific gene expression was conserved in old DR mice which had to get prolonged life span and decreased lymphoma incidence. Thereby, gene expression of endogenous retroviruses appears to change during aging and to be modifiable by life-prolonging DR. It may be therefore used as a biomaker of aging in mice. Humans are known to have similar gene elements like MuLV. The present findings demonstrate a possibility of application of endogenous gene expressions to the epidemiology of aging.

Demonstration of a new mouse mammary tumor virus locus in the genome of the mammary tumor phone strain SHN mouse

Until this report, there have been no detailed analyses of the mouse mammary tumor virus (MMTV) loci in the mammary tumor prone strain SHN. Using a probe, which hybridizes to the env sequence of MMTV, Southern blotting of genomic DNA from the brain after digestion with EcoRI revealed 5 endogenous proviruses: Mtv-1 (4.5kb), Mtv-2 (11 kb), Mtv-8 (6.7kb), Mtv-17 (8.3kb) and a newly-found 6.5-kb fragment. F1-hybrid mice (C3H/He female x SHN male) also possessed the 6.5-kb fragment. Thus, we conclude that the 6.5-kb fragment is unique to SHN mice. Genomic DNA from mammary tumors of SHN mice showed MMTV insertions, suggesting that activation of an oncogene(s) occurred in this strain.

Energy restriction suppresses microsomal Ca2+-ATPase activities in various organs in C57BL/6 female mice in both euthermic and torpor states

C57BL/6 female mice were fed a daily control diet (n = 5, 3.9 g/day, 95 kcal/week) or ER diet (n = 5, 2.3 g/day, 48 kcal/week) at 1800 h from 6 weeks of age. Telemetry, conducted at 6 months of age, confirmed that all ER mice entered daily torpor (core body temperature less than 31 degrees C) for 6.63 +/- 2.34 h/day while control mice were euthermic (> 35 degrees C). In vitro activities of microsomal Ca(2+)-ATPase were determined in the brain, liver, salivary gland and kidney from these mice at 6 months of age. Assays were performed at three incubation temperatures of 37 degrees C, 31 degrees C and 25 degrees C. In assays at 37 degrees C, the activities of Ca(2+)-ATPase in the brain and salivary gland from ER mice were lower than those in corresponding organs from control mice. The suppression became profound as the incubation temperature decreased. On the other hand, at 37 degrees C Ca(2+)-ATPase activities in the liver and kidney from ER mice were not lower than those in corresponding organs from control mice, but decreased significantly at low temperatures. Microsomal Ca(2+)-ATPase activities thus appeared to be reduced in ER mice, although it remains unknown whether the present results represent reduced in vivo capacities to regulate cytosolic Ca2+ concentrations.

O,O,S-Trimethyl phosphorothioate induces hypothermia in Fischer 344 rats in a manner dependent on both doses and housing temperatures.

We explored the effects of O,O,S-trimethyl phosphorothioate (OOS-TMP) on body temperatures in Fischer 344 female rats. The 7-day LD50 p.o. for Fischer 344 female rats was found to be 11.8 mg/kg. OOS-TMP induced long-lasting (more than 48 h) and extensive hypothermia at doses > 14 mg/kg at a typical laboratory temperature (22° C) while it produced typical symptoms at 10 mg/kg without hypothermia. In contrast, pair-fed (to 20 mg/kg rats) rats (n=4) did not become hypothermic, negating any role of hypophagia in OOS-TMP associated hypothermia. We next investigated the effects of housing temperatures on toxicities at a LD50 dose (12 mg/kg). At 30° C (n=11) and 22° C (n=13), rats did not have hypothermic bouts but at 15° C, eight out of ten rats had. Evidence that changes of housing temperatures neither modified clinical symptoms nor changed mortality rates discards a possibility of hypothermia being involved in delayed toxicity. A novel result of the present study suggests that thermoregulation may be heavily impaired by a special class of organophosphorus compounds.

Electrophysiological evidence of an increase in cold tolerance of cardiac muscles in mice after energy restriction

Life-prolonging energy restriction (ER) has been known to extend longevity. The heart was selected as the target organ of ER and the electrophysiological properties of ER on the heart were investigated. Action potential parameters were measured on ventricular papillary muscles of C57BL/6 mice (2-6 months of age). Resting membrane potential (Rm) did not change even when the temperature was lowered to 20 degrees C in ER mice (-67.5 +/- 0.8 mV), however, the membrane was depolarized in the control (-61.1 +/- 1.1 mV). Action potential duration measured at 30 and 50% repolarization was significantly prolonged in ER mice at 20-35 degrees C. Ouabain (10 microM) decreased Rm in ER mice at 20 degrees C (-68.6 +/- 1.0 to -63.6 +/- 0.8 mV), but failed to decrease Rm in the control (-60.6 +/- 1.8 to -62.1 +/- 1.2 mV). There were no significant differences in extracted Na, K-ATPase activity or affinity and binding capacity of ouabain between ER and control hearts. These results indicate that in ER mice the lack of effect of temperature on Rm was not due to a change in the physicochemical properties of Na, K-ATPase. The present study collectively suggests that ER increases cold tolerance in the heart of mice.