[3H]d-Aspartic acid release in brain slices of adult and aged fischer 344 rats

Alterations in glutamate content and uptake have been reported to occur in aged animals. The present studies used [³H]d-Aspartic acid ([³H]-D-ASP) release as a marker for glutamate neurotransmission. Frequency dependent [³H]-D-ASP release was measured in adult (8 month) and aged (28–30 month) Fischer 344 rats. Relatively high stimulation frequencies (>10 Hz) were required to induce [³H]-D-ASP release in both adult and aged F344 rats in temporal cortex and hippocampus. In both brain areas aged animals showed significantly more [³H]-D-ASP release than adult animals Kainic acid 1 mM failed to induce the release of [³H]-D-ASP in either temporal cortex or hippocampus. Omega conotoxin GVIA (5×10^–9M) a N and L type voltage sensitive calcium channel antagonist failed to inhibit [³H]-D-ASP stimulated release. These results demonstrate an increase in [³H]-D-ASP release in aged compared to adult F344 rats. The data also suggest a novel calcium channel may be involved in [³H]-D-ASP release.     

Dietary lithium during development: Changes in amino acid levels,ionic content,and [3H]spiperone binding in the brain of rats

We found that chronic lithium diet affects the sensitivity of neuroleptic receptors and the content of amino acids in the brain, and that the changes in adult animals differ from those in young rats. Pregnant rats were kept on lithium diet (pellets with 0.21% Li2CO3 and 0.21% NaCl) during the gestation period and the offspring were kept on lithium for six weeks after delivery. Control rats were kept on normal diet under identical conditions. In corpus striatum and cerebral cortex of lithium-treated young rats a reduction in apparent dissociation constant and no change in (3H)spiperone total binding sites were found, suggesting a sensitization of the neuroleptic receptor; this result was unlike that obtained with adult lithium-treated rats, where the total number of binding sites was decreased. The lithium content of brain was very high (2.32 meq/kg of wet weight), whereas in the serum only 0.75 meq/l was recorded. K+ and Na+ levels increased by 20% and 9% respectively in the brain and remained at normal levels in the serum. Analysis of free amino acids in the cerebral cortex, midbrain, and cerebellum showed increases in GABA and glycine levels in all three regions, a significant increase in taurine in midbrain, and an increase in lysine in cerebral cortex and cerebellum. The results indicate that the effect of chronic dietary lithium given during pregnancy on the neuroleptic receptor in young rats is different from that in adult animals. It produces an increase in the number of the neuroleptic receptor sites instead of the decline in the number of binding sites found in adult rats. It remains to be established whether this effect is related more to the age of the animal tested or to the stage of development of the CNS at which the lithium was administered.     

Oxidative stress in head trauma in aging

Oxidative damage is proposed as a key mediator of exacerbated morphological responses and deficits in behavioral recovery in aged subjects with traumatic brain injury (TBI). In the present study, we show exacerbated loss of tissue in middle aged (12 months) and aged (22 months) Fisher-344 rats compared to young animals (3 months) subjected to moderate TBI. Analysis of 4-hydroxynonenal (4-HNE) and acrolein, neurotoxic by-products of lipid peroxidation, shows significant (P < 0.05) age-dependent increases in ipsilateral (IP) hippocampus 1 and 7 days post injury. In IP cortex, 4-HNE was significantly elevated 1 day post injury in all age groups, and both 4-HNE and acrolein were elevated in middle aged and aged animals 7 days post injury. Comparison of antioxidant enzyme activities shows significant (P < 0.05) age-dependent decreases of manganese superoxide dismutase in IP hippocampus and cortex 1 and 7 days post injury. Glutathione reductase activity also showed an age-dependent decrease. Overall, our data show increased levels of oxidative damage, diminished antioxidant capacities, and increased tissue loss in TBI in aging.     

3H-Dihydromorphine binding in brain regions of young and aged rats

Opiate receptor-ligand binding was investigated in brains of young and aged female F344 rats. A significant reduction in the density of binding sites for ³H-dihydromorphine was observed in the thalamus and midbrain of aged rats. Evidence of two binding sites was observed in the anterior cortex of young rats, whereas aged rats exhibited only a single site. The occurrence of pituitary tumors in the old female rats did not affect ³H-dihydromorphine binding. The highest density of dihydromorphine binding sites was found in the diencephalon, and the lowest density in the hippocampus. Receptor densities in the anterior cortex, striatum, amygdala, frontal poles and midbrain were intermediate, and few, if any, high affinity binding sites for dihydromorphine were found in the pons-medulla or posterior cortex.     

Maternal Undernutrition During Lactation: Effect on Amino Acids in Brain Regions of Offspring

Sprague-Dawley dams were fed either a protein-calorie deficient or control diet from day 5 to day 21 after parturition. The concentrations of seven amino acids (aspartate, glutamate, gamma-aminobutyric acid, glycine, glutamine, serine, and taurine) were determined in brain regions from 17-day-old undernourished offspring and from 35-day-old rehabilitated rats. The brain regions examined were the cortex, cerebellum, corpus striatum, hippocampus, hypothalamus, brainstem, and midbrain. At 17 days of age, taurine was the amino acid with the highest concentration, whereas at 35 days glutamate had the highest concentration. This change was due to the fact that the concentration of taurine decreased significantly in all brain regions between 17 and 35 days, whereas the concentration of glutamate remained high or increased somewhat in all brain regions except the hypothalamus and brainstem. When the age-matched offspring of control and undernourished rats were compared, several interesting and significant differences were found. The concentrations of glutamate and aspartate were significantly lower (decreased 16-34%) in the cerebellum, brainstem, cortex, and midbrain in 17-day-old undernourished rats. The aspartate level was also significantly decreased in the corpus striatum and hypothalamus in 17-day-old offspring. However, the deficiencies of aspartate and glutamate were transient and reversible. In contrast, the concentration of taurine was increased in the hypothalamus (31%) and hippocampus (12-33%) at both 17 and 35 days of age and in the midbrain (17%) at 17 days. Other transient abnormalities in amino acid levels were found in undernourished offspring. The results of these experiments suggest that undernutrition during lactation causes delayed CNS development, which is manifested in altered concentrations of the neurotransmitters aspartate, glutamate, and taurine.     

Immunoreactive 7B2 concentrations in rats with various endocrine conditions

Changes in 7B2 immunoreactivity in the pituitary as well as in the other brain regions and gut after various endocrine situations were investigated. Gonadectomy and neonatal monosodium glutamate (MSG) treatment resulted in an appreciable increase in the pituitary 7B2 concentration, though 7B2 content in the MSG treated pituitary was not significantly different when calculation was performed on a per pituitary gland basis. The 7B2 concentration in the cerebellum, midbrain and cortex in thyroxine treated rats showed a significant increase, which might indicate possible thyroid hormone involvement in 7B2 metabolism in the brain. The pituitary 7B2 concentration during the estrous cycle did not change significantly. These results suggest that pituitary 7B2 may correlate to the pituitary gonadotropins and that brain 7B2 content may be modulated by thyroid hormones.     

Effect of substance P on the reproduction of memory engrams and on the amino acid composition of brain structures in rats with experimental neurosis

Formation of experimental neurosis in rats was accompanied in 64% of animals by development of amnesia of conditioned reaction of passive avoidance. Disturbance of mnestic processes was manifested by a change in free amino acids pool including the acids with neurotransmitter properties (GABA, glutamate, glycine). An increase of GABA and glycine content was found in the frontal cortex and an increase of glutamate and GABA--in the hippocampus and striate body. Substance P (125 mkg/kg) administered intraperitoneally against the background of a developed neurosis, produced in 80% of cases an antiamnestic action, accompanied by a statistically significant decrease of GABA content in the frontal cortex, hippocampus and midbrain, and an increase of glutamate in the midbrain. The level of taurine decreased in the frontal cortex, hippocampus and striate body, and increased in the midbrain. Threonine content increased in the striate body and midbrain; there was an increase of taurine, serine and glycine in the midbrain and of glycine in the striate body.     

Age related changes in weight and the concentrations of zinc and copper in the brain of the adult rat

Two groups of adult male rats aged 15 weeks and 49 weeks, 15 rats in each group, were analysed for the concentrations of the trace elements zinc (Zn) and copper (Cu) in serum, liver, kidney, and five parts of the brain (cortex, corpus striatum, hippocampus, midbrain + medulla, and cerebellum). All organs increased in weight from 15 weeks to 49 weeks. In all parts of the brain, except for corpus striatum, there was a significant increase of the weights. The dry weight (% of wet) increased in all parts of the brain. In serum, the Zn and Cu concentrations increased from 15 weeks to 49 weeks. In the liver, both concentrations decreased and in the kidney the concentrations increased with increasing age. The Zn concentrations increased in cortex and corpus striatum and decreased in cerebellum and hippocampus. The Cu levels increased in all parts of the brain with the largest changes in corpus striatum. For rats aged 49 weeks, a significant correlation was found between the Cu concentrations of corpus striatum or midbrain + medulla and the fluid consumption. The findings of the present study reveal a dynamic age-related pattern of changes in the concentrations of Zn and Cu in different organs of the adult rat. This stresses the need of age-matching as an important control in experiment studies.     

Deficits in Sustained Attention and Changes in Dopaminergic Protein Levels following Exposure to Proton Radiation Are Related to Basal Dopaminergic Function

The current report assessed the effects of low-level proton irradiation in inbred adult male Fischer 344 and Lewis rats performing an analog of the human Psychomotor Vigilance Test (PVT), commonly utilized as an object risk assessment tool to quantify fatigue and sustained attention in laboratory, clinical, and operational settings. These strains were used to determine if genetic differences in dopaminergic function would impact radiation-induced deficits in sustained attention. Exposure to head-only proton irradiation (25 or 100 cGy) disrupted rPVT performance in a strain-specific manner, with 25 cGy-exposed Fischer 344 rats displaying the most severe deficits in sustained attention (i.e., decreased accuracy and increased premature responding); Lewis rats did not display behavioral deficits following radiation. Fischer 344 rats displayed greater tyrosine hydroxylase and dopamine transporter levels in the frontal cortex compared to the Lewis rats, even though radiation exposure increased both of these proteins in the Lewis rats only. Tyrosine hydroxylase was decreased in the parietal cortex of both rat strains following radiation exposure, regardless of proton dose. Strain-specific cytokine changes were also found in the frontal cortex, with the Lewis rats displaying increased levels of putative neurotrophic cytokines (e.g., CNTF). These data support the hypothesis that basal dopaminergic function impacts the severity of radiation-induced deficits in sustained attention.     

Effect of Acute Stress on Hippocampal Glutamate Levels and Spectrin Proteolysis in Young and Aged Rats

Abstract: Aging in rats is associated with a loss of hippocampal neurons, which may contribute to age-related cognitive deficits. Several lines of evidence suggest that stress and glucocorticoids may contribute to age-related declines in hippocampal neuronal number. Excitatory amino acids (EAAs) have been implicated in the glucocorticoid endangerment and stress-induced morphological changes of hippocampal neurons of young rats. Previously, we have reported that acute immobilization stress can increase extracellular concentrations of the endogenous excitatory amino acid, glutamate, in the hippocampus. The present study examined the effect of an acute bout of immobilization stress on glutamate levels in the hippocampus and medial prefrontal cortex of young (3–4-month) and aged (22–24-month) Fischer 344 rats. In addition, the effect of stress on spectrin proteolysis in these two brain regions was also examined. Spectrin is a cytoskeleton protein that contributes to neuronal integrity and proteolysis of this protein has been proposed as an important component of EAA-induced neuronal death. There was no difference in basal glutamate levels between young and old rats in the hippocampus or medial prefrontal cortex. During the period of restraint stress a modest increase in glutamate levels in the hippocampus of young and aged rats was observed. After the termination of the stress procedure, hippocampal glutamate concentrations continued to rise in the aged rats, reaching a level approximately five times higher than the young rats, and remained elevated for at least 2 h after the termination of the stress. A similar pattern was also observed in the medial prefrontal cortex with an augmented post-stress-induced glutamate response observed in the aged rats. There was no increase in spectrin proteolysis in the hippocampus or medial prefrontal cortex of young or aged rats after stress or under basal nonstress conditions. The enhanced poststress glutamate response in the aged rats may contribute to the increased sensitivity of aged rats to neurotoxic insults.     

Effect of acetyl-l-carnitine on extracellular amino acid levels in vivo in rat brain regions

Acetyl-l-carnitine (ALCAR) was found to have beneficial effects in senile patients. In recent years many of its effects on the nervous system have been examined, but its mechanism(s) of action remains to be elucidated. We previously reported that it causes release of dopamine in the striatum. In the present paper we report that ALCAR, when administered at intracerebral sites via microdialysis, stimulates the release of amino acids in a concentration-dependent and regionally heterogeneous manner. The effect was strong in the striatum and cerebellum, less so in the frontal cortex, and weak in the thalamus. Seven amino acids were measured: the increase in the level of aspartate, glutamate, and taurine was substantial, and the increase in the level of glycine, serine, threonine, alanine, and glutamine in the microdialysate was minor. The stimulatory effect of ALCAR on the release of amino acids in the striatum was inhibited by the muscarinic antagonist atropine, but was not inhibited by the nicotinic antagonist mecamylamine. The effect of ALCAR on the levels of most of the amino acids tested was independent of the presence of Ca²⁺ in the perfused. These results indicate that ALCAR, when administered intracerebrally at fairly high concentrations, can affect the level and the release not only of such neurotransmitters as acetylcholine and dopamine, but also of amino acids. The mechanism of action of ALCAR on the release of cerebral amino acids may involve the participation of muscarinic receptors or may be mediated through the release of dopamine, but the lack of Ca²⁺ dependence indicates a release from the cytoplasmic amino acid pool, possibly through the effect of ALCAR on cell membrane permeability.     

Possible role of glutamate, aspartate, glutamine, GABA or taurine on cadmium toxicity on the hypothalamic pituitary axis activity in adult male rats

This work was designed to evaluate the possible changes in glutamate, aspartate, glutamine, GABA and taurine within various hypothalamic areas the striatum and prefrontal cortex after oral cadmium exposure in adult male rats, and if these changes are related to pituitary hormone secretion. The contents of glutamine, glutamate, aspartate, GABA and taurine in the median eminence, anterior, mediobasal and posterior hypothalamus, and in prefrontal cortex in adult male rats exposed to 272.7 mol l^–1 of cadmium chloride (CdCl₂) in the drinking water for one month. Cadmium diminished the content of glutamine, glutamate and aspartate in anterior hypothalamus as compared to the values found in the untreated group. Besides, there is a decrease in the content of glutamate, aspartate and taurine in the prefrontal cortex. The amino acids studied did not change in median eminence, mediobasal and posterior hypothalamus or the striatum by cadmium treatment. Plasma prolactin and LH levels decreased in rats exposed to the metal. These results suggest that (1) cadmium differentially affects amino acid content within the brain region studied and (2) the inhibitory effect of cadmium on prolactin and LH secretion may be partially explained by a decrease in the content of both glutamate and aspartate in anterior hypothalamus, but not through changes in GABA and taurine.     

Regional differences in glutaminase activation by phosphate and calcium in rat brain: Impairment in aged rats and implications for regional glutaminase isozymes

Regional regulation of glutaminase by phosphate and calcium was examined in the temporal cortex (TCX), striatum (STR) and hippocampus (HIPP) from adult and aged male F344 rats. Phosphate-dependent glutaminase activity in adult rats was significantly lower (35–43%) in the HIPP (100 and 150 mM) and STR (150 mM) compared to PAG activity in the TCX. Phosphate activation in aged rats was 50–60% lower in the HIPP at concentrations greater than 25 mM compared to the aged TCX or STR. PAG activity in the TCX and STR was unaffected by age, but was significantly reduced (30–50%) in the HIPP from aged rats at phosphate concentrations of 25 mM and greater when compared to adult rats. In adult rats at concentrations of CaCl₂ above 1 mM, PAG activity was significantly lower (60–75%) in the STR and HIPP when compared to the TCX. In aged rats, PAG activity (1 mM CaCl₂) in the HIPP was significantly less (50%) than STR PAG activity in aged rats. Diminished PAG activity was seen only in the TCX (2.5 mM; 32%), and the HIPP (0.5 mM; 25% and 1 mM; 38%) at higher calcium concentrations compared to adult. Phosphate-independent calcium activation of PAG occurred in the HIPP but not in either the TCX or the STR. Addition of phosphate resulted in a synergistic activation of PAG in the STR and TCX, but not in the HIPP. These findings suggest that PAG is regionally regulated by phosphate and calcium, and this regulation is impaired in aged rats. These data also support the hypothesis that isozymes of PAG exist with different regulatory properties.Abbreviation PAG Phosphate-activated glutaminase - L-glutamine amidohydrolase EC 3.5.1.2 - TCX temporal cortex - STR striatum - HIPP hippocampus - F344 Fischer-344 rat     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Neurotoxin effects on oxytocin, vasopressin and somatostatin in discrete rat brain areas

Monosodium-L-Glutamate (MSG) produces lesions to monoaminergic and peptidergic neurons in several brain areas. The present study examined the effect of neonatal MSG treatment on oxytocin (OXY), arginine-vasopressin (AVP) and somatostatin (SRIF) concentrations in several discrete brain areas of adult rats. OXY increased in the suprachiasmatic and arcuate nuclei and median eminence (ME) and decreased in the paraventricular nucleus of MSG-treated rats. MSG treatment caused AVP to increase in the arcuate nucleus and ME and decrease in the supraoptic nucleus. SRIF decreased following neonatal MSG treatment in both the ME and neurointermediate pituitary lobe. The results demonstrate that the effects of neonatal MSG treatment on neuropeptide content are not just limited to the arcuate nucleus. Furthermore, taken together with previous results, the data suggest that these changes may be indicative of functional deficits in the neuronal activity of some of these peptidergic neurons which, in turn, may be responsible for the abnormal secretion of several pituitary hormones observed in MSG-treated animals.     

Expression of alpha-synuclein in different brain parts of adult and aged rats.

The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.     

Effects of aging on adipose resistance artery vasoconstriction: possible implications for orthostatic blood pressure regulation.

The purpose of this investigation was to determine mean arterial pressure (MAP) and regional vascular conductance responses in young and aged Fisher-344 rats during orthostatic stress, i.e., 70 degrees head-up tilt (HUT). Both groups demonstrated directionally different changes in MAP during HUT (young, 7% increase; aged, 7% decrease). Vascular conductance during HUT in young rats decreased in most tissues but largely remained unchanged in the aged animals. Based on the higher vascular conductance of white adipose tissue from aged rats during HUT, resistance arteries from white visceral fat were isolated and studied in vitro. There was diminished maximal vasoconstriction to phenylephrine and norepinephrine (NE: young, 42 +/- 5%; old, 18 +/- 6%) in adipose resistance arteries from aged rats. These results demonstrate that aging reduces the ability to maintain MAP during orthostatic stress, and this is associated with a diminished vasoconstriction of adipose resistance arteries.     

Age-related alteration of poly(ADP-ribose) polymerase activity in different parts of the brain

Poly(ADP-ribose) polymerase (PARP) is a conserved enzyme involved in the regulation of DNA repair and genome stability. The role of PARP during aging is not well known. In this study PARP activity was investigated in nuclear fractions from hippocampus, cerebellum, and cerebral cortex of adult (4 months), old adult (14 months) and aged (24-27 months) rats. Concomitantly, the free radical evoked lipid peroxidation was estimated as thiobarbituric acid reactive substances (TBARS). The specific activity of PARP in adult brain was about 25, 21 and 16 pmol/mg protein per min in hippocampus, cerebellum and cerebral cortex, respectively. The enzyme activity was higher in all investigated parts of the brain of old adults. In aged animals PARP activity was lower in hippocampus by about 50%, and was unchanged in cerebral cortex and in cerebellum comparing to adult rats. The concentration of TBARS was the same in all parts of the brain and remained unchanged during aging. There is no direct correlation between PARP activity and free radical evoked lipid peroxidation during brain aging. The lowered enzyme activity in aged hippocampus may decrease DNA repair capacity which subsequently may be responsible for the higher vulnerability of hippocampal neurons to different toxic insults.     

Histological changes and neurotransmitter levels three months following perinatal asphyxia in the rat.

The involvement of excitatory amino acids (EAA) in the pathogenesis of hypoxic-ischemic states is well-documented. Information on the role of overexcitation by EAA in perinatalasphyxia (PA), however, is limited and data from adult models cannot be directly extrapolated to immature systems. Moreover, most adult models of ischemia are representing stroke rather than PA. We decided to study long term effects in a non-invasive rat model of PA resembling the clinical situation three months following the asphyctic insult. Morphometry on Nissl - stained sections was used to determine neuronal death in frontal cortex, striatum, hippocampus CA1, hypothalamus and cerebellum L1, and the amino acids glutamate, glutamine, aspartate, GABA, taurine, arginine as well as histamine, serotonin and 5-hydroxy-indoleacetic acid were determined in several brain regions and areas. Morphometry revealed that neuronal loss was present in the hippocampal area CA1 in all groups with PA and that morphological alterations were significantly higher in the cerebellar granular layer. The prominent light microscopical finding in all areas of asphyctic rats studied was decreased Nissl-staining, suggesting decreased cellular RNA levels. Glutamate, aspartate and glutamine were significantly elevated in the hypothalamus of asphyctic rats probably indicating overstimulation by EAA. Excitotoxicity in this area would be compatible with findings of emotional / behavioral deficits observed in a parallel study in our model of PA. Our observations point to and may help to explain behavioral and emotional deficits in Man with a history of perinatal asphyxia.     

Biological aging does not lead to the accumulation of oxidized Cu,Zn-superoxide dismutase in the liver of F344 rats

Cu,Zn-Superoxide dismutase (SOD) was isolated from the liver of 3-, 12-, and 26-month-old Fisher 344 (F344) rats. Specific activity and metal content of the enzyme, purified by ion-exchange and size-exclusion chromatography, did not significantly change with age. Electrospray ionization-mass spectrometry and amino acid analysis of Cu,Zn-SOD apoprotein, further purified by reverse-phase HPLC, showed neither significant loss of amino acids nor accumulation of oxidized isoforms with age. When bovine Cu,Zn-SOD, oxidized with H(2)O(2) in vitro, was added to rat liver homogenate, we reisolated circa 70% of the oxidized bovine Cu,Zn-SOD together with the rat isoform, showing that oxidized Cu,Zn-SOD can be recovered from tissue homogenate. Therefore, our data do not confirm an earlier hypothesis that oxidatively modified Cu,Zn-SOD protein accumulates in the liver of aged F344 rats.