bluuebunny/crossref_metadata_2025 huggingface dataset:
Haematological, hepatic and renal alterations after repeated oral or intraperitoneal administration of monoisoamyl DMSA. I. Changes in male rats
Authors: Mehta Ashish , Kannan G. M. , Dube S. N. , Pant B. P. , Pant S. C. , Flora S. J. S.
Published: November 2002

Monoisoamyl 2,3‐dimercaptosuccinic acid (MiADMSA), a vicinal thiol chelator, is gaining recognition recently as a better chelator than meso 2,3‐dimercaptosuccinic acid (DMSA) in decreasing heavy metal burden in tissues because of its lipophilic character. There is, however, little information available on the toxicological properties of this chelator after repeated administration in animals. In the present study, we investigated the dose‐dependent effect of MiADMSA on various biochemical parameters suggestive of alterations in haem biosynthesis and hepatic, renal and brain oxidative stress after 21 days of repeated intraperitoneal (i.p.) or oral (p.o.) administration to rats. The concentration of essential metals in blood and soft tissues was determined along with histopathological observations of hepatic and renal tissues.

The results suggest that MiADMSA administration had no effect on blood δ‐aminolevulinic acid dehydratase activity. However, an increase in zinc protoporphyrin and a decrease in haemoglobin levels were noted in animals given MiADMSA i.p. A moderate increase in serum alkaline phosphatase suggested mild hepatotoxicity at the highest dose (100 mg kg−1, i.p.). This was confirmed by histopathological examinations, which identified basophilic stippling, granulation of the cytoplasm, haemorrhage and congestion. At the highest dose, levels of hepatic thiobarbituric acid reactive substance and oxidized glutathione were increased above those of control values. Levels of hepatic reduced glutathione were decreased. Taken together, these observations point to oxidative stress. In animals administered MiADMSA i.p. there was an increase in the brain malondialdehyde levels at the two higher doses (50 and 100 mg kg−1). Essential metal status revealed a significant effect of MiADMSA (p.o.) in increasing blood zinc while significantly decreasing the kidney zinc level. The most significant adverse effect of MiADMSA was on copper concentration, which showed significant depletion from almost all major organs. Magnesium levels in blood decreased but increased in liver of MiADMSA‐administered rats. Histopathological observations of liver and kidneys suggest few moderate lesions.

It can be concluded that repeated administration of MiADMSA is compromised with some mild toxic effect, particularly the loss of copper. The effects during oral administration are comparatively less pronounced than by the i.p. route. Copyright © 2002 John Wiley & Sons, Ltd.
Responses in the respiratory tract of rats following exposure to sulphuric acid aerosols for 5 or 28 days
Authors: Kilgour Joanne D. , Foster John , Soames Anthony , Farrar David G. , Hext Paul M.
Published: November 2002

Sulphuric acid mists have been classified by the International Agency for Research on Cancer as being carcinogenic to humans based on epidemiological findings of respiratory tract tumours. To determine if early changes in the respiratory tract following exposure to sulphuric acid (H2SO4) aerosols are consistent with the possible development of tumours after extended periods of exposure, groups of female rats were exposed to respirable aerosols of H2SO4 at target concentrations of 0, 0.2, 1.0 or 5.0 mg m−3 for 6 h per day for either 5 days or for 5 days a week over a 28‐day period. Additional groups exposed to 0 or 5.0 mg m−3 over the 28‐day period were retained after exposure for 4 or 8 weeks to assess recovery. Histopathological examinations and quantitative cell proliferation measurements were conducted on the nasal passages, larynx and lung. Achieved concentrations were 0.3, 1.38 and 5.52 mg m−3 H2SO4. Histological and cell proliferative changes were confined to the larynx and no effects were seen in the nasal passages or lungs. At the two highest concentrations, squamous metaplasia accompanied by significant cell proliferation was apparent after 5 and 28 days of exposure and there was a reduction in the severity of the pathological changes following the recovery periods. No effects were seen at 0.3 mg m−3 after 5 days of exposure and only minimal metaplastic change was seen after 28 days in a few animals and was not accompanied by cell proliferation. The toxicological relevance of these findings is discussed. Copyright © 2002 John Wiley & Sons, Ltd.
Differential stimulation of IgE production, STAT activation and cytokine and CD86 expression by 2,4‐dinitrochlorobenzene and trimellitic anhydride
Authors: Guo T. L. , Zhang X. L. , Leffel E. K. , Peachee V. L. , Karrow N. A. , Germolec D. R. , White K. L.
Published: November 2002

It has been reported that dermal exposure to trimellitic anhydride (TMA, 50%), a respiratory allergen, induced greater production of serum IgE and expression of Th2 cytokines than 2,4‐dinitrochlorobenzene (DNCB, 1%), a potent contact sensitizer, in female BALB/C mice. To determine if there is any strain difference, four strains (B6C3F1, C57BL/6, BDF1 and BALB/C) of female mice were employed in this study to compare the differential effects of these chemicals on the hypersensitivity responses. Serum IgE levels were increased in TMA‐treated B6C3F1, C57BL/6 and BDF1 mice when compared with the DNCB treatment and vehicle controls; in contrast, no difference was observed between TMA‐ and DNCB‐treated BALB/C mice, although both chemicals induced greater IgE production than vehicle controls. In vitro expression of interleukin 4 (IL‐4) and IL‐13 mRNA by overnight concanavalin A (ConA)‐stimulated draining lymph node cells was enhanced following in vivo treatment with TMA but not with DNCB in the B6C3F1, C57BL/6 and BDF1 mice. In contrast, TMA and DNCB induced similar levels of IL‐4 and IL‐13 mRNA in the BALB/C mice. The IL‐4 protein levels in the supernatants of overnight ConA‐treated draining lymph node cells were also increased in TMA‐treated B6C3F1 and C57BL/6 mice when compared with the DNCB treatment and vehicle controls. Further mechanistic evaluation in the B6C3F1 mice indicated that the activation of STAT6 but not STAT4 by ConA plus IL‐2‐treated draining lymph node cells was increased in TMA‐ but not DNCB‐treated mice when compared with the vehicle controls. Furthermore, surface expression of B7.2 (CD86) by B cells was increased in both TMA‐ and DNCB‐treated B6C3F1 mice when compared with the vehicles; however, greater B7.2 expression was observed in TMA‐treated compared with DNCB‐treated. Overall, these results demonstrate that a similar pattern of IgE and cytokine production was observed in these strains of mice except for BALB/C. Furthermore, differential activation of STAT6 and expression of CD86 following exposure to TMA and DNCB may contribute to the differential production of IgE and cytokines. Copyright © 2002 John Wiley & Sons, Ltd.
Ultrastructural examination of rabbit aortic wall following high‐fat diet feeding and selenium supplementation: a transmission electron microscopy study
Authors: Mehta U. , Kang B. P. S. , Kukreja R. S. , Bansal M. P.
Published: November 2002

Experiments were carried out to examine the changes occurring in the wall of rabbit aortae following high‐fat diet (HFD) feeding as well as HFD + selenium supplementation. Male New Zealand White rabbits were divided into three groups—control, HFD‐fed and HFD + Se supplementation—and were treated for three months. The study depicted that levels of serum total cholesterol and triglycerides were markedly increased in the HFD‐fed group as compared with control animals. However, in the HFD + Se‐fed group, these levels were markedly suppressed vis‐à‐vis animals fed on HFD only. Development of atherogenic and atheromatic plaques has been shown at the light microscopy level in HFD‐fed rabbits, whereas these developments were not visible in the HFD + Se‐fed rabbits. Transmission electron microscopy findings indicated altered ultrastructure in the endothelial cells of the intimal layer as well as smooth‐muscle cells of the medial layer in HFD‐fed animals. However, these findings indicated normal ultrastructure in most of the cells, with little ultrastructural alterations from animals supplemented with Se along with HFD feeding. The study on the whole depicted the ability of Se to inhibit the onset of progression of aortic disease and hence has relevance to its therapeutic potential. Copyright © 2002 John Wiley & Sons, Ltd.
Comparative effect of dietary administration of Lathyrus sativus pulse on behaviour, neurotransmitter receptors and membrane permeability in rats and guinea pigs
Authors: Amba Archana , Seth Kavita , Ali Mohamed , Das Mukul , Agarwal A. K. , Khanna Subhash K. , Seth P. K.
Published: November 2002

Neurolathyrism, an upper motor neuron disease, has been thought to be caused by long‐term dietary consumption of lathyrus pulse, which contains the toxin β‐N‐oxalyl‐L‐α,β‐diaminopropionic acid. Earlier behavioural studies employing oral feeding of lathyrus pulse to animals has been conducted without evaluating the biochemical toxicity potential. In the present investigation the effect of dietary feeding of 10%, 50% and 80% lathyrus pulse to rats and guinea pigs for 3 months on neurobehavioural parameters, including locomotor activity, inclined plain test and neurotoxicological parameters such as neurotransmitter receptor binding, Ca2+ influx and membrane fluidity, was investigated. Exposure of 50% low and high toxin lathyrus to rats did not cause any significant change in locomotor activity, whereas guinea pigs at the same dosage regimen of high toxin lathyrus showed significant lowering of inclined plain test scores. Furthermore, studies of neuroreceptor binding in rats fed 50% low and high toxin lathyrus showed significant changes in glutamate, dopamine and muscarinic receptors, whereas the benzodiazepine receptor elicited no change. Guinea pigs, on the other hand, fed 50% and 80% lathyrus in the diet showed significant changes in glutamate, dopamine, muscarinic and benzodiazepine receptors. Interestingly, significant elevation in intracellular calcium with a concomitant increase in membrane fluidity was observed in rats (50% low and high toxin) and guinea pigs (50% and 80%) fed a lathyrus diet. These results indicate that although both species (rats and guinea pigs) are susceptible to neurochemical changes on exposure to lathyrus, locomotor changes are only noticed in guinea pigs. Thus, guinea pigs may be more prone to lathyrus toxicity and may serve as a sensitive animal model compared with rats. Copyright © 2002 John Wiley & Sons, Ltd.
Lack of changes in brain muscarinic receptor and motor activity of mice after neonatal inhalation exposure to d‐allethrin
Authors: Tsuji Ryozo , Kobayashi Kumiko , Ikeda Maya , Yoshioka Takafumi , Yamada Tomoya , Seki Takaki , Okuno Yasuyoshi , Nakatsuka Iwao , Tsuruo Yoshihiro , Kishioka Shiroh
Published: November 2002

Synthetic pyrethroids are among the most common pesticides and insecticides currently in worldwide use. Eriksson and co‐workers postulated that oral exposure of mice to pyrethroids during a neonatal brain growth spurt induces permanent disturbance in the cerebral muscarinic cholinergic receptor (MAChR) and behaviour. However, the scientific basis for these phenomena is now under discussion. The present study was performed to determine whether the experimental findings of Eriksson's study could be reproduced in newborn mice by inhalation. Male and female NMRI mice were exposed to d‐allethrin by whole‐body inhalation for 6 h per day between postnatal days 10 and 16. Actual concentrations of d‐allethrin were 0.43, 1.35, 3.49 and 74.2 mg m−3 (equivalent to 0.70, 2.2, 5.7 and 120.2 mg kg−1 day−1, respectively), and the mass median aerodynamic diameter and geometric log‐standard deviation of mist particles ranged from 2.58 to 2.98 µm and from 1.58 to 2.09 µm for all groups, respectively. The highest exposure level in the present study (74.2 mg m−3) was ca. 13 000 times as high as the concentration used in practice. The MAChR in the three brain areas (cortex, hippocampus and striatum) and motor activity were examined at the ages of 17 days and 4 months. In addition, a water‐maze test was performed at the age of 11 months. There was no systemic toxicity interfering with the interpretation of assay results. The neonatal exposure to d‐allethrin by inhalation did not induce effects either on the brain MAChR density and motor activity at 17 days and 4 months or on performance in the learning/memory test at the age of 11 months. The effects of allethrins on developmental neurotoxicity that Eriksson and co‐workers reported previously were not reproduced in the present study. Copyright © 2002 John Wiley & Sons, Ltd.
Dose–response relationship between total cadmium intake calculated from the cadmium concentration in rice collected from each household of farmers and renal dysfunction in inhabitants of the Jinzu River basin, Japan
Authors: Kobayashi Etsuko , Okubo Yasushi , Suwazono Yasushi , Kido Teruhiko , Nogawa Koji
Published: November 2002

The association between total cadmium (Cd) intake and abnormal urinary findings was investigated in the Cd‐polluted Jinzu River basin. In 1967 and 1968 the most systematic and large‐scale health examinations were conducted among the entire population aged over 30 years of this region. We performed this study by targeting the subjects participating in the 1967 health survey conducted mainly in the heavily polluted area. From subjects who had eaten household rice of known Cd concentration, 1075 inhabitants who had either resided in their current household since birth or who had moved there from a non‐polluted area (group A), and 780 inhabitants who had resided in the current household since birth (group B), were selected as the target population. The total Cd intake for each person was calculated from the Cd dose ingested from rice and other foods. Logistic regression analysis was performed using the prevalence of abnormal urinary findings (proteinuria, glucosuria and proteinuria with glucosuria) as the criterion variable and the total Cd intake and age as explanatory variables. In subjects of groups A and B the odds ratios became higher as the dose of total Cd intake increased. Odds ratios in subjects of group A were statistically significant except for glucosuria of men and proteinuria of women. In subjects of group B the odds ratios were also significant for proteinuria + glucosuria of men and glucosuria of women.

It was demonstrated that the greater the increase in total Cd intake, the greater the increase in abnormal urinary findings in the Jinzu River basin, and the association of the two factors was very close. Copyright © 2002 John Wiley & Sons, Ltd.
Distinguishing animal subsets in toxicokinetic studies: comparison of non‐linear mixed effects modelling with non‐compartmental methods
Authors: Hing Jeremy P. , Woolfrey Steven G. , Greenslade Dennis , Wright Peter M. C.
Published: November 2002

The purpose of this study was to compare the ability of non‐compartmental analysis and compartmental mixed effects modelling (MEM) to determine the existence and magnitude of exposure differences (i.e. exposure ratio estimates) between subsets of animals during destructive toxicokinetic studies. Data from five toxicokinetic studies of an experimental compound were analysed using a linear trapezoidal calculation of the area under the curve (non‐compartmental analysis) or modelled using MEM. With the non‐compartmental method the Bailer–Satterthwaite approximation was used to construct confidence intervals around the exposure estimates of each subset of animals and these were used to determine if exposure differed between the subsets. The MEM analyses were performed on the full datasets and on datasets with arbitrary reductions in the number of animal replicates. With MEM, additional model parameters were used to differentiate between subsets of animals, and were incorporated only if they were justified statistically. Estimates of the existence and magnitude of exposure differences between animal subsets were similar with the two techniques. The MEM analyses were influenced only marginally by substantial reductions in the number of animals studied and were less compromised by extremely limited or unbalanced data. These analyses show that MEM and non‐compartmental methods are similarly effective at detecting exposure differences between animal subsets in toxicokinetic studies. Estimates provided by both methods were influenced by the degree of variance in the data. These results support the proposition that it may be possible to reduce the number of animals employed in toxicokinetic studies if MEM is used. Copyright © 2002 John Wiley & Sons, Ltd.
Novel method for detection of ex vivo tumor necrosis factor α production by monocytes
Authors: Suzuki Kikumi , Koyama Takatoshi , Kobayashi Shizuko , Kobayashi Koji , Inagaki Koji , Abe Yoshiko , Kuriyama Kiyoshi , Shiba Kiyoko
Published: November 2002

Tumor necrosis factor α (TNF‐α) has come into recent focus as a proinflammatory cytokine derived from monocytes/macrophages. We developed a novel system (the SEK‐5001 system) for measurement of ex vivo production of TNF‐α stimulated by lipopolysaccharide (LPS) as a marker of immune function. In the present study, we evaluated the performance of this system as a diagnostic tool. Furthermore, we compared TNF‐α levels with data from other immune function tests, including lymphocyte blast formation test and differential leukocyte counts. Incubation of whole blood with a stimulation of low‐dose LPS (100 EU/mL blood) for 4hr at 37°C gave acceptable results. After incubation, plasma TNF‐α levels were determined by enzyme‐linked immunosorbent assay (ELISA). The specificity, reproducibility, and recovery of the SEK‐5001 system were excellent. No correlation between TNF‐α levels and total leukocyte counts was found. Lymphocyte blast formation test and monocyte counts, however, were correlated with TNF‐α levels in blood from patients with hematological malignancy and aplastic anemia before treatment. This assay system may potentially be clinically applicable to assess in vivo immune function. J. Clin. Lab. Anal. 16:273–278, 2002. © 2002 Wiley‐Liss, Inc.
Clinical application of the serum 1,5‐anhydroglucitol assay method using glucose 3‐dehydrogenase
Authors: Hamafuji Tetsuro , Tsugawa Wakako , Sode Koji
Published: November 2002

We attempted to develop a novel serum 1,5‐anhydroglucitol (1,5‐AG) assay kit using glucose 3‐dehydrogenase (G3DH) from Halomonas sp. α‐15 strain. The major advantages of this method are that the 1,5‐AG detection requires a very small amount of G3DH, and the enzyme catalyzes a simple reaction. The analytical performances were acceptable for clinical use operated with a clinical automatic analyzer. The correlation with a commercial assay kit against sera of healthy volunteers was y=0.975x+0.008, r=0.993, Sy∣x=1.32 μg/mL. However, sham‐negative specimens were observed in the validation of this method using specimens from hospital patients. J. Clin. Lab. Anal. 16:299–303, 2002. © 2002 Wiley‐Liss, Inc.
Changes in the mannan binding lectin (MBL) concentration in human milk during lactation
Authors: Trégoat Virginie , Montagne Paul , Béné Marie‐Christine , Faure Gilbert
Published: November 2002

The mannan binding lectin (MBL) activates the complement system by the lectin pathway after the recognition of some structural motifs (saccharides) present on the surface of microorganisms. MBL has been mostly identified and quantified in human serum by ELISA or microparticle immunonephelometry assays. This article reports the MBL levels as assessed by a microparticle immunonephelometric assay in 76 human milk samples. Immunonephelometry was performed using skim‐milk samples diluted 20 times over a calibration range of 0.07–4.82 mg/L. MBL is indeed present in human milk and its concentration decreases significantly during development from colostrum (0.55±0.09 mg/L) to transitional (0.18±0.02 mg/L) and mature milk (0.17±0.02 mg/L). This innate molecule may be involved in the primary defenses of the mammary gland and the neonate, whose immune system is immature. The high levels observed during the first days of lactation support the hypothesis that this molecule plays a key role in limiting the colonization of the newborn gut by pathogens. J. Clin. Lab. Anal. 16:304–307, 2002. © 2002 Wiley‐Liss, Inc.
Drug–herb interactions: unexpected suppression of free Danshen concentrations by salicylate
Authors: Gupta Deepali , Jalali Mehri , Wells Alice , Dasgupta Amitava
Published: November 2002

The general population of the U.S. uses over‐the‐counter herbal medicines. Danshen is a Chinese herbal product used for the treatment of cardiovascular disease. In a previous study we showed that Danshen has significant digoxin‐like immunoreactivity, and we used this parameter to monitor total and free Danshen activities in sera (10). In this report we demonstrated strong protein binding of Danshen (50–70%), and we also identified albumin as the major serum protein that binds Danshen. Because salicylate, which is also strongly bound to albumin, is a widely used over‐the‐counter medicine in the U.S., we studied Danshen–salicylate interaction in vitro. We observed no significant change in free Danshen concentrations as measured by free‐digoxin‐like activity when salicylate concentrations were subtherapeutic (≤100 μg/mL). With therapeutic concentrations of salicylate (≥150 μg/mL), the free Danshen concentrations significantly decreased from the control. On the other hand, Danshen can displace salicylate from protein binding, thereby increasing the free salicylate concentration. We conclude that salicylate in therapeutic concentration can significantly decrease free Danshen concentrations, and Danshen can displace salicylate. J. Clin. Lab. Anal. 16:290–294, 2002. © 2002 Wiley‐Liss, Inc.
Preparation and characterization of anti‐anti‐idiotypic monoclonal antibody (“Ab1‐like Ab3”) in relation to carcinoembryonic antigen
Authors: Tsujisaki Masayuki , Masuya Jiro , Okada Yoshimasa , Jinnohara Tsuneharu , Sasaki Shigeru , Yachi Akira , Imai Kohzoh
Published: November 2002

In order to analyze the epitope structure of carcinoembryonic antigen (CEA) and the idiotype network system, seven anti‐idiotypic monoclonal antibodies (anti‐Id MoAbs) were generated from a BALB/c mouse immunized with anti‐CEA MoAb P1‐356, which recognized a synthetic peptide P1 of CEA, domain I. These MoAbs were divided into four groups. The anti‐Id MoAbs specifically reacted with MoAb P1‐356, but not with any MoAb, and inhibited the binding of MoAb P1‐356 to CEA, indicating that all of these anti‐Id MoAbs recognized private idiotopes at the combining sites of MoAb P1‐356. Polyclonal anti‐anti‐idiotypic antiserum (Ab3) generated with anti‐Id MoAbs M315 (Ab2), blocked the binding of MoAb P1‐356 (Ab1) to CEA and reacted with CEA(Ag) and synthetic peptide P1. The analysis of serological assays suggested that it contains “Ab1‐like Ab3.” Therefore, we prepared anti‐anti‐Id MoAbs using anti‐Id MoAb M315. Among 13 candidates, anti‐anti‐Id MoAb 11B2 was selected, because it competed with MoAb P1‐356 (Ab1) binding to CEA. A direct binding assay using MoAb11B2 showed that it reacted with purified CEA and with CEA synthetic peptide P1. In addition, MoAb 11B2 (Ab3) reacted with both CEA‐producing cultured cells and colonic cancerous tissues in immunostaining. These results indicate that anti‐anti‐Id MoAb 11B2 is “Ab1‐like Ab3.” Therefore, it is suggested that anti‐Id MoAb M315 bears an “internal image” of the MoAb P1‐356‐defined epitope on CEA. J. Clin. Lab. Anal. 16:279–289, 2002. © 2002 Wiley‐Liss, Inc.
Effect of dietary restriction beyond middle age: Accumulation of altered proteins and protein degradation
Authors: Takahashi Ryoya , Goto Sataro
Published: November 2002

Dietary restriction (DR) from weaning or young adult stages in rodents throughout their usual life span has been shown to prolong longevity and lower or delay (or both) the occurrence of many late‐onset diseases, but the effects of DR when performed at middle age or later have not been well investigated. We have studied the effect of DR initiated late in life on altered proteins and protein degradation. DR initiated in 23.5‐month‐old mice and continued for 2 months reduced the amount of altered proteins and shortened the half‐life of proteins, including oxidatively modified ones. Furthermore, our more recent studies in rats demonstrated that DR initiated later in life (26.5 months old) restored the activities of proteasomes that have been implicated in removal of altered proteins. Thus, DR initiated even relatively late in life appears to have beneficial effects, restoring an animal's youthful condition in terms of the age‐related accumulation of altered proteins. Microsc. Res. Tech. 59:278–281, 2002. © 2002 Wiley‐Liss, Inc.
Impact of aging and life‐long calorie restriction on expression of apoptosis‐related genes in male F344 rat liver
Authors: Ando Kohei , Higami Yoshikazu , Tsuchiya Tomoshi , Kanematsu Takashi , Shimokawa Isao
Published: November 2002

Aging enhances apoptosis of hepatocytes under normal physiological conditions and increases the susceptibility to apoptosis of hepatocytes, whereas chronic calorie restriction (CR) suppresses the age‐enhanced susceptibility to apoptosis. To clarify the subcellular mechanisms of age‐associated dysregulation of apoptosis and the effects of CR, we analyzed the expression of genes promoting apoptosis (p53, Fas receptor, Fas ligand, TNF receptor 1, TNFα, Bax, TGFβ1) and genes preventing apoptosis (Bcl‐2 and Bcl‐XL) in the livers of 3‐, 6‐, 15‐, and 24‐month‐old male F344 rats that were either fed ad libitum or subjected to a 30% reduction in food intake (CR). After the age of 6 months, expression of p53, Fas receptor, Fas ligand, and TNFα mRNAs was up‐regulated with aging. CR suppressed this age‐enhanced p53 and Fas receptor mRNA expression, but expression of the other genes was not altered significantly by aging or CR. Expression of Fas receptor in hepatocytes, as detected immunohistochemically, increased with age, but CR suppressed age‐accelerated Fas receptor expression. Our findings suggest that TNF ligand/TNF receptor family signaling, particularly Fas receptor expression, is important in age‐ and CR‐modulated apoptosis of hepatocytes. Hepatocytes that were immunoreactive for p53 had slightly increased with aging, suggesting that p53 may mediate the age‐enhanced up‐regulation of Fas receptor in hepatocytes. Microsc. Res. Tech. 59:293–300, 2002. © 2002 Wiley‐Liss, Inc.
Effects of food restriction on motoneuronal loss with advancing age in the rat
Authors: Kanda Kenro
Published: November 2002

The effects of life‐long food restriction on motoneuronal cell death with advancing age was studied in male Fischer rats, which had access to food only 3 days a week after weaning (FR rats). Motoneurons innervating the medial gastrocnemius muscle were labeled with retrogradely transported HRP. The number of labeled motoneurons in FR rats and rats fed ad libitum (AL rats) was similar at the age of 16 months (131.8 ± 1.7 for FR rats vs. 133.8 ± 4.5 for AL rats). However, at 28 months of age, AL rats had less labeled motoneurons compared to FR rats (117.0 ± 2.4 for FR rats vs. 124.3 ± 7.0 for FR rats). The number of type I muscle fibers in the medial gastrocnemius muscle increased significantly in AL rats during the period from 16 to 28 months of age, which might reflect motor unit reorganization following retraction of axons and/or death of innervating motoneurons. FR rats did not show statistically significant alteration. These findings were also true for the data compiled from several different experiments including those conducted for primarily different purposes in our laboratory. The results suggest that life‐long food restriction retards motoneuronal cell death occurring with advancing age. Microsc. Res. Tech. 59:301–305, 2002. © 2002 Wiley‐Liss, Inc.
Effects of caloric restriction on development of the proximal growth plate and metaphysis of the caput femoris in spontaneously hypertensive rats: Microscopic and computer‐assisted image analyses
Authors: Kawahara Toshio , Shimokawa Isao , Tomita Masato , Hirano Toru , Shindo Hiroyuki
Published: November 2002

We previously demonstrated that caloric restriction (CR) reduced the prevalence of osteonecrosis in caput femoris of spontaneously hypertensive rats (SHR), a model of human Perthes' disease. The effects of CR on the development and pathology in the proximal femoral growth plate (GP) and adjacent structures in SHR were investigated by morphometric and computer‐assisted image analyses. From 6 weeks of age, the food intake of SHR was restricted to 65% of the mean intake of ad libitum fed control SHR (SHR‐AL). Wistar Kyoto rats (WKY), from which the SHR strain was isolated, fed ad libitum were also included as controls. CR reduced prevalence of chondromucinous degeneration and dysarray of cartilage cell columns in the GP, becoming prevalent between 10 and 20 weeks in the SHR‐AL group, attaining the same levels of the WKY group. Thicknesses of non‐calcifying cell columns in the GP were greater in the SHR‐AL than WKY group; CR slightly reduced the thickness, but incompletely. Thicknesses of calcifying cell columns did not significantly differ among the three groups. CR decreased volume density and mean thicknesses of the trabecular bone in areas adjacent to GPs, and was greater in the SHR‐AL than the WKY group. The present morphologic analysis suggested that CR ameliorates dysplastic changes of trabecular bones in areas adjacent to the GP, rather than modulating the ossification process in the GP. The CR paradigm might give insight into the pathogenesis of, and a therapeutic strategy for, human Perthes' disease. Microsc. Res. Tech. 59:306–312, 2002. © 2002 Wiley‐Liss, Inc.
Characterization of gene expression profile associated with energy restriction‐induced cold tolerance of heart
Authors: Wada Yasuhiko , Kayo Tsuyoshi , Koizumi Akio
Published: November 2002

Hypothermia is known to be a common feature of energy restriction (ER) and essential for a life‐prolonging effect of ER. The heart is sensitive to hypothermia, but the heart in ER mice acquires some adaptation to hypothermia. The aim of the present study was to characterize the gene expression profile associated with ER‐induced cold resistance of heart. We analyzed the expression of heart mRNA from ER (200 kJ/week) or control (400 kJ/week) B6 11‐month‐old male mice using cDNA array membranes including 588 genes. Eighty‐eight out of 588 genes were expressed in the heart. mRNAs increased by ER were glutathion S‐transferase Mu1, transcriptional factor 1 for heat shock gene (HSF1), and fetal myosin alkali light chain genes. mRNA decreased by ER were seven genes in four categories: (1) cell cycle or apoptosis‐related proteins (cyclin G and nucleoside diphosphate kinase B); (2) stress response proteins (oxidative stress‐induced protein); (3) DNA repair proteins (protein involved in DNA double‐strand break repair, Rad23 UV excision repair protein homologue and ubiquitin‐conjugating enzyme); and (4) cell‐surface antigens (lamimin receptor 1). These data suggest that the heart of ER mice adapts to hypothermia involving heat shock proteins and their transcriptional factors and by changing structure and property of myofibrils. It is also suggested that ER induces protection against oxidative stress and inhibits cell proliferation of “nonmuscle cells” in the heart. Gene expression analysis using cDNA array was useful for screening genes associated with ER‐induced cold adaptation. Microsc. Res. Tech. 59:313–316, 2002. © 2002 Wiley‐Liss, Inc.
Anti‐aging effects of caloric restriction: Involvement of neuroendocrine adaptation by peripheral signaling
Authors: Chiba Takuya , Yamaza Haruyoshi , Higami Yoshikazu , Shimokawa Isao
Published: November 2002

Many hormonal signals from peripheral tissues contribute to the regulation of energy homeostasis and food intake. These regulators including leptin, insulin, and ghrelin, modulate the orexigenic and anorexigenic neuropeptide expression in hypothalamic nuclei. The anti‐aging effects of caloric restriction have been explained from an evolutional viewpoint of the adaptive response of the neuroendocrine and metabolic response systems to maximize survival during periods of food shortage. In organisms, excess energy is stored in adipose tissues as a triglyceride preparation for such survival situations. Adipose tissue has recently been recognized as an endocrine organ, and leptin, as secreted by adipocyte, seems to be an especially important factor for the adaptive response to fasting and neuroendocrine alterations under caloric restriction. In this review, we discuss the potential involvement of neuroendocrine modulators in longevity and the anti‐aging effects of caloric restriction. Microsc. Res. Tech. 59:317–324, 2002. © 2002 Wiley‐Liss, Inc.
Lifespan extension by caloric restriction: An aspect of energy metabolism
Authors: Yamaza Haruyoshi , Chiba Takuya , Higami Yoshikazu , Shimokawa Isao
Published: November 2002

Caloric restriction (CR) may retard aging processes and extend lifespan in organisms by altering energy‐metabolic pathways. In CR rodents, glucose influx into tissues is not reduced, as compared with control animals fed ad libitum (AL), although plasma concentrations of glucose and insulin are lower. Gene expression profiles in rodents have suggested that CR promotes gluconeogenesis and fatty acid biosynthesis in skeletal muscle. In the liver, CR promotes gluconeogenesis but decreases fatty acid synthesis and glycolysis. In lower organisms such as yeasts and nematodes, incomplete blocks in steps of insulin/insulin‐like growth factor‐1 (IGF‐1) signal pathway extend lifespan. The life‐prolonging effect of CR in yeasts requires NPT1 and SIR2 genes, both of which relate to sensing energy status and silencing genes. These findings stress the substantial role of energy metabolism on CR. Future studies on metabolic adaptation and gene silencing with regard to lower caloric intake will be warranted to understand the mechanisms of the anti‐aging and life‐prolonging effects of CR. Microsc. Res. Tech. 59:325–330, 2002. © 2002 Wiley‐Liss, Inc.
Effects of aging and caloric restriction on the gene expression of Foxo1, 3, and 4 (FKHR, FKHRL1, and AFX) in the rat skeletal muscles
Authors: Furuyama Tatsuo , Yamashita Hitoshi , Kitayama Kazuko , Higami Yoshikazu , Shimokawa Isao , Mori Nozomu
Published: November 2002

In C. elegans, insulin‐like hormone signal pathway plays a significant role in longevity. In particular, daf‐16 gene product is indispensable factor for this lifespan‐extension. This signal pathway is critical for dauer formation, which is a similar state to hibernation in mammals. We examined the expression level of mammalian daf‐16 homologues, Foxo 1,3, and 4 (FKHR, FKHRL1, and AFX) mRNAs in the rat skeletal muscles during aging and in 30% caloric restricted of ad libitum fed. The expression level of AFX mRNA was significantly higher at 6 and 12 months than at 3 and 26 months, and FKHRL1 expression was significantly higher at 6 months than at 3 and 26 months but FKHR expression showed no significant change with age. We observed a characteristic expression of AFX and FKHR mRNAs to be significantly higher in the second day in caloric restriction by every‐other‐day feeding than in ad libitum fed. This suggests that caloric restriction may increase the expression of FKHR‐family genes and prevent the aging process in the skeletal muscles. Microsc. Res. Tech. 59:331–334, 2002. © 2002 Wiley‐Liss, Inc.
Caloric restriction and aging in primates: Relevance to humans and possible CR mimetics
Authors: Lane Mark A. , Mattison Julie , Ingram Donald K. , Roth George S.
Published: November 2002

For nearly 70 years it has been recognized that reduction in caloric intake by 30–40% from ad libitum levels leads to a significant extension of mean and maximal lifespan in a variety of short‐lived species. This effect of caloric restriction (CR) on lifespan has been reported in nearly all species tested and has been reproduced hundreds of times under a variety of different laboratory conditions. In addition to prolonging lifespan, CR also prevents or delays the onset of age‐related disease and maintains many physiological functions at more youthful levels. Studies in longer‐lived species, specifically rhesus and squirrel monkeys, have been underway since the late 1980s. The studies in nonhuman primates are beginning to yield valuable information suggesting that the effect of CR on aging is universal across species and that this nutritional paradigm will have similar effects in humans. Even if CR can be shown to impact upon human aging, it is unlikely that most people will be able to maintain the strict dietary control required for this regimen. Thus, elucidation of the biological mechanisms of CR and development of alternative strategies to yield similar benefits is of primary importance. CR mimetics, or interventions that “mimic” certain protective effects of CR, may represent one such alternative strategy. Microsc. Res. Tech. 59:335–338, 2002. © 2002 Wiley‐Liss, Inc.
Diffusion parameters in the striatum of rats with 6‐hydroxydopamine‐induced lesions and with fetal mesencephalic grafts
Authors: Reum T. , Olshausen F. , Mazel T. , Voříšek I. , Morgenstern R. , Syková E.
Published: November 2002

Functional recovery after transplantation of dopaminergic cells into the lesioned striatum is dependent on widespread diffusion of the transmitter released by the graft. In the present study, we investigated the diffusion parameters of the extracellular space in the striatum of control, 6‐hydroxydopamine‐lesioned, intrastriatally grafted, and sham‐grafted rats in vivo. We used two types of grafts—single macrografts or multiple micrografts. The real‐time iontophoretic tetramethylammonium method enabled us to extract three extracellular space diffusion parameters: volume fraction, α, tortuosity, λ, and nonspecific uptake of tetramethylammonium, k′. Compared with controls (α = 0.19, λ = 1.59), in lesioned animals both α and λ were lower (α = 0.14, λ = 1.50). α and λ were increased inside macro‐and micrografts, where α = 0.24 and λ = 1.80, and in sham‐grafted areas, where α = 0.24 and λ = 1.72. In regions outside the grafts (α = 0.15, λ = 1.51) or in sham grafts (α = 0.14, λ = 1.49), the values of α and λ were similar to the values observed in lesioned striatum. Nonspecific uptake (k′) did not differ among the groups. Our results show that, compared with control, α and λ were decreased in dopamine‐depleted areas and increased in areas with grafts. Multiple but smaller graft deposits, in contrast to their enlarged capability for dopaminergic reinnervation, impair the conditions for diffusion and extrasynaptic transmission in a larger area of the striatum than do single macrografts, presumably because of more extensive tissue damage, cell loss, and astrogliosis. © 2002 Wiley‐Liss, Inc.
The DNA damaging agent etoposide activates a cell survival pathway involving α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors and mitogen‐activated protein kinases in hippocampal neurons
Authors: Lu Chengbiao , Fu Weiming , Zhao Daohong , Mattson Mark P.
Published: November 2002

Etoposide, an inhibitor of topoisomerase II that induces DNA damage and can trigger cell death, is used as a chemotherapeutic agent. Because chemotherapies can result in neurological complications and because DNA damage in neurons is implicated in the pathogenesis of several neurodegenerative disorders, we studied the effects of etoposide on cultured hippocampal neurons. We found that etoposide induces neuronal apoptosis and that, prior to the cell death commitment point, there is an increase in whole‐cell α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐induced current but no change in N‐methyl‐D‐aspartate (NMDA)‐induced current. Associated with the increase in AMPA‐induced current was an increase in the amounts of AMPA receptor subunits GluR1 and GluR4, whereas levels of the NMDA receptor subunit NR1 were unaffected by etoposide. AMPA receptor activation can result in excitotoxic cell death but can also activate signaling pathways that promote synaptic plasticity and cell survival. We found that etoposide increases the activation of p42 and p44 mitogen‐activated protein (MAP) kinases, and that activation of the MAP kinases by etoposide requires AMPA receptor activation. Pharmacological blockade of AMPA receptors and p42/p44 MAP kinases, but not of NMDA receptors, exacerbated etoposide‐induced cell death. These findings suggest that, although etoposide is neurotoxic, it also activates a cell survival pathway involving AMPA receptor‐mediated activation of p42/p44 MAP kinases. Agents that selectively inhibit the cell life or death pathways triggered by DNA damage may prove useful in the settings of cancer and neurodegenerative disorders, respectively. Published 2002 Wiley‐Liss, Inc.
Multiple aspects of homocysteine neurotoxicity: Glutamate excitotoxicity, kinase hyperactivation and DNA damage
Authors: Ho Pei I. , Ortiz Daniela , Rogers Eugene , Shea Thomas B.
Published: November 2002

Homocysteine (HC) is a neurotoxic amino acid that accumulates in several neurological disorders including Alzheimer's disease (AD). We examined the consequences of treatment of cultured murine cortical neurons with HC. Homocysteine‐induced increases in cytosolic calcium, reactive oxygen species, phospho‐tau immunoreactivity and externalized phosphatidyl serine (indicative of apoptosis). Homocysteine‐induced calcium influx through NMDA channel activation, which stimulated glutamate excitotoxicity, as evidenced by treatment with antagonists of the NMDA channel and metabotropic glutamate receptors, respectively. The NMDA channel antagonist MK‐801 reduced tau phosphorylation but not apoptosis after HC treatment, suggesting that HC‐mediated apoptosis was not due to calcium influx. Apoptosis after HC treatment was reduced by co‐treatment with 3‐aminobenazmidine (3ab), an inhibitor of poly‐ADP‐ribosome polymerase (PARP), consistent with previous reports that ATP depletion by PARP‐mediated repair of DNA strand breakage mediated HC‐induced apoptosis. Treatment with 3ab did not reduce tau phosphorylation, however, therefore hyperphosphorylation of tau may not contribute to HC‐induced apoptosis under these conditions. Inhibition of mitogen‐activated protein kinase by co‐treatment with the kinase inhibitor PD98059 inhibited tau phosphorylation but not apoptosis after HC treatment. HC accumulation reduces cellular levels of S‐adenosyl methionine (SAM); co‐treatment with SAM reduced apoptosis, suggesting that inhibition of critical methylation reactions may mediate HC‐induced apoptosis. These findings indicate that HC compromises neuronal homeostasis by multiple, divergent routes. © 2002 Wiley‐Liss, Inc.
Oligodendrogenesis is differentially regulated in gray and white matter of jimpy mice
Authors: Baracskay Karen L. , Duchala Cynthia S. , Miller Robert H. , Macklin Wendy B. , Trapp Bruce D.
Published: November 2002

The factors that regulate oligodendrogenesis have been studied extensively in optic nerve, where oligodendrocyte production and myelination quickly follow colonization of the nerve by progenitor cells. In contrast, oligodendrocyte production in the cerebral cortex begins approximately 1 week after progenitor cell colonization and continues for 3–4 weeks. This and other observations raise the possibility that oligodendrogenesis is regulated by different mechanisms in white and gray matter. The present study examined oligodendrocyte production in the developing cerebral cortex of jimpy (jp) and jimpymsd (msd) mice, which exhibit hypomyelination and oligodendrocyte death due to mutations in and toxic accumulations of proteolipid protein, the major structural protein of CNS myelin. Proliferation of oligodendrocyte progenitors and production of myelinating oligodendrocytes was reduced in jp cerebral cortex when compared to wild‐type (wt) and msd mice. The incidence of oligodendrocyte cell death was similar in jp and msd cortex, but total dying oligodendrocytes were greater in msd. We confirm previous reports of increased oligodendrocyte production in white matter of both jp and msd mice. The jp mutation, therefore, reduces oligodendrocyte production in cerebral cortex but not in white matter. These data provide additional evidence that oligodendrogenesis is differentially regulated in white matter and gray matter and implicate PLP/DM20 as a modulator of these differences. © 2002 Wiley‐Liss, Inc.
Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis
Authors: Belletti Silvana , Orlandini Guido , Vettori Maria Vittoria , Mutti Antonio , Uggeri Jacopo , Scandroglio Renato , Alinovi Rossella , Gatti Rita
Published: November 2002

Organic mercury is a well‐known neurotoxicant although its mechanism of action has not been fully clarified. In addition to a direct effect on neurons, much experimental evidence supports an involvement of the glial component. We assessed methylmercury hydroxide (MeHgOH) toxicity in a glial model, C6 glioma cells, exposed in the 10−5–10−8 M range. The time course of the effects was studied by time‐lapse confocal microscopy and supplemented with biochemical data. We have monitored cell viability and proliferation rate, reactive oxygen species (ROS), mitochondrial transmembrane potential, DNA oxidation, energetic metabolism and modalities of cell death. The earliest effect was a measurable ROS generation followed by oxidative DNA damage paralleled by a partial mitochondrial depolarization. The effect on cell viability was dose dependent. TUNEL, caspase activity and real‐time morphological observation of calcein‐loaded cells showed that apoptosis was the only detectable mode of cell death within this concentration range. N‐acetyl‐cysteine (NAC) or reduced glutathione (GSH) completely prevent the apoptotic effect of MeHgOH. The lowest effective MeHgOH concentration was 10−7 M for ROS and DNA OH‐adducts generation. The effect of submicromolar concentrations of MeHgOH on C6 cells could be relevant in the developmental neurotoxicity caused by low dose, long‐term exposures, such as those of food origin. In addition, we have shown that the same concentrations are effective in the induction of DNA oxidative damage, with further potential pathogenetic implications. © 2002 Wiley‐Liss, Inc.
JNK3 contributes to c‐jun induction and apoptosis in 4‐hydroxynonenal‐treated sympathetic neurons
Authors: Bruckner Shane R. , Estus Steven
Published: November 2002

4‐Hydroxynoneal (HNE), an end product of lipid peroxidation, induces apoptosis in many cell types, including neural cells. HNE toxicity is often accompanied by activation of the c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) pathway. Here we have evaluated the hypothesis that the primary JNK associated with neurons, JNK3, contributes to HNE‐induced neuronal apoptosis. First, we demonstrate that HNE induces caspase‐dependent apoptosis in sympathetic neurons. Second, we show that HNE‐induced c‐Jun phosphorylation and c‐jun induction are attenuated in JNK3‐deficient neurons. Third, we show that HNE neurotoxicity is significantly inhibited by JNK3 deficiency. In summary, these results indicate that JNK3 plays a critical role in HNE‐induced c‐Jun activation and apoptosis in sympathetic neurons. © 2002 Wiley‐Liss, Inc.