Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age.

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)+ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.

8OHdG levels in brain do not indicate oxidative DNA damage in Alzheimer's disease.

Accumulation of oxidative DNA damage has been proposed to underlie aging and neurodegenerative diseases such as Alzheimer's Disease (AD). The DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG) is considered a good indicator of oxidative DNA damage. To investigate whether this type of DNA damage is involved in AD etiology, 8OHdG levels were determined in postmortem human brain tissue of controls and AD patients (in frontal, occipital, and temporal cortex and in hippocampal tissue). Parametric studies in rat revealed no influences of postmortem delay, repeated freezing/thawing or storage time. In human brain, approximately two 8OHdG molecules were present per 10(5) 2'-deoxyguanosines. In AD patients and controls, 8OHdG-levels were not related to age, sex, or brain region. Also, no differences were found between controls and AD patients. It was concluded that 8OHdG in nuclear DNA, although present throughout the brain in fairly high amounts, does not accumulate with age, nor does it appear to be involved in AD. More detailed studies are required to extend this conclusion to other types of oxidative damage.

Influence of age-related changes in rodent liver morphology and physiology on drug metabolism--a review.

Age-related changes in weight, morphology and physiology of the rodent liver influencing hepatic drug metabolism are reviewed. Next to the changes in liver weight/body weight ratio with age, the spontaneous occurrence of neoplastic and non-neoplastic lesions may be of particular importance. In addition, the decrease in liver blood blow with age diminishes the biotransformation capacity of the total liver. However, the albumin concentration in plasma and drug uptake do not play important roles, since they are unchanged or only slightly lower in old rats or mice. Drugs are generally metabolized by the liver in two phases: the so-called phase I and phase II metabolism. For most drugs, the phase I reaction is an oxidation. This reaction is catalyzed by cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase. In microsomes, a decrease with age is generally observed in the cytochrome P-450 concentration and the NADPH-cytochrome c reductase activity, while there is no change in cytochrome b5. In addition, most microsomal drug-metabolizing enzymes decrease with age in male rats but not in females. The changes in enzyme activities in the male and female mouse are more complex. In fact, increases, decreases and no changes were found. Important phase II reactions are glutathione conjugation and glucuronidation; changes in both reactions with age seem to be of minor importance. Studies with hepatocytes isolated from male rats of different ages reveal that the monooxygenase system mediated metabolism of digitoxin and aflatoxin B1 decreases with age. It can be concluded that the observed decrease in the functional capacity of the monooxygenase system greatly determines the decrease in drug metabolism with age. However, it should always be kept in mind that, among others, the age-related changes in drug metabolism in rats are strongly sex dependent, which is not the case in man. Therefore, caution should be exercised in transferring these data to the human situation.

Pharmacokinetics of hexobarbital in young and old rats.

Hexobarbital is a drug widely used to study the capacity of the liver to metabolize drugs. The pharmacokinetics of hexobarbital in 3- and 30-month-old male BN/BiRij rats were studied. The half-life of hexobarbital in 30-month-old rats (39.9 +/- 4.1 min) was significantly higher than that of 3-month-old ones (21.3 +/- 3.8 min). The volume of distribution (ml.kg-1 body weight) did not change with age. The intrinsic clearance, expressed as ml.min-1.kg-1 body weight, of hexobarbital in 30-month-old rats (20.2 +/- 6.6) was half that of the 3-month-old ones (39.5 +/- 7.6). Further studies will be performed to investigate the effect of age on the intrinsic clearance of S(+)- and R(-)- hexobarbital.

The effect of age on antipyrine metabolism by liver microsomes isolated from phenobarbital-treated rats.

A study was performed to test the hypothesis that ageing influences the activities of diverse forms or populations of cytochrome P-450 isoenzymes in different ways. The formation of antipyrine metabolites in induced rats is mediated by such different forms or populations of cytochrome P-450 isoenzymes. To test the hypothesis, the formation rates of antipyrine metabolites by liver microsomes isolated from phenobarbital-treated rats of different ages was determined. After phenobarbital induction in vitro, the maximal velocity for norantipyrine formation decreased from 12 to 24 months and then showed a tendency to increase with age. Hydroxymethylantipyrine formation did not change with age. 4-Hydroxyantipyrine increased between 3 and 12 months and remained constant afterwards. This is in agreement with data obtained in vivo in uninduced male BN/BiRij rats. It can be concluded that age does indeed influence the activities of different forms or populations of the cytochrome P-450 isoenzymes in different ways. Consequently, determining the overall clearance of antipyrine, which is metabolized by several isoenzymes, especially in the induced situation, is not to be recommended for measuring the activity of cytochrome P-450 isoenzymes as a function of age.

The effect of age on the oxidative metabolism of antipyrine by uninduced microsomal fractions of rat liver.

The influence of ageing on the metabolism of antipyrine by different forms of cytochrome P-450 was studied in vitro, by measuring the formation of antipyrine metabolites by microsomes isolated from untreated rats, which were grouped into 5 different ages. Km, Vmax and Vmax/Km values were determined for the metabolites: 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA) and 4-hydroxyantipyrine (OHA). Km values for all metabolites ranged from 2.0 to 5.0 mM and Vmax values from 1.84 to 3.66 nmol/min per mg. The Vmax/Km ratios varied from 0.65 to 1.73 microliters/min per mg. With respect to the Km values, a decrease for HMA from 12 to 24 months, no change for OHA and an increase for NORA from 3 to 30 months, was observed. The Vmax values showed an increase for HMA from 3 to 24 months, a decrease for OHA from 12 to 30 months and no change for NORA. The absolute Vmax/Km ratios for all metabolites showed no significant changes during ageing. However, regarding the relative Vmax/Km ratios, an increase for HMA from 3 to 24 months, a decrease for OHA from 12 to 35 months and also a decrease for NORA from 3 to 24 months, was observed. It is concluded that ageing influenced the Km, Vmax and Km/Vmax ratio of the different P-450 isoenzymes involved in antipyrine metabolism, in different ways. However, the age-related changes are modest (10-100%), especially for the Vmax/Km ratio.

Respiratory activities of hepatocytes isolated from rats of various ages. A brief note.

The ratio between the oxygen consumption of isolated hepatocytes in the presence of an inhibitor of oxidative phosphorylation (oligomycin, atractyloside) and in the presence of an uncoupler (dinitrophenol) is used as an estimation of the respiratory control ratio of mitochondria inside the isolated liver parenchymal cell. No significant age-related decline in the functional integrity of the mitochondria could be detected.

The effect of age on protein synthesis by isolated liver parenchymal cells.

The protein synthesizing capacity of liver parenchymal cells isolated from 3-, 12-, 24-, 31- and 36-month-old rats was determined by the incorporation of 14C-leucine. Conditions for optimum protein synthesis included the use of an enriched medium (modified Waymouth's MB 752/1) and cell suspension concentrations ranging from 0.25 to 4 X 10(6) cells/ml medium. The cells were incubated with a dose of 6 micronmol leucine/ml medium for 2 h at 37 degrees C under an atmosphere of 95% O2 and 5% CO2. With parenchymal cells isolated from 3-month-old rats, a leucine incorporation rate of 14.4 nmol leucine/h/10(6) cells was found. The capacity of the parenchymal cells to synthesize protein decreased between 3 and 12 months, remained constant between 12 and 24 months and increased between 24 and 26 months. Degradation of newly synthesized proteins or reutilization of 14C-leucine did not occur during the incubation period. The ratio between albumin and total protein synthesis as a function of age was determined. This ratio did not change between 3 and 24 months but there was a significant increase between 24 and 36 months. The increase in total protein synthesis in late age may be due to a compensation by the liver for a more pronounced proteinuria, increased proteolysis or an accumulation of "altered" proteins.

Sex and strain dependency of age-related changes in protein synthesis of isolated rat hepatocytes.

In previous studies, a decrease in protein synthesis by hepatocytes isolated from female WAG/Rij rats was observed in the first year of life, while an increase was seen in advanced age. In the present study, no change in protein synthesis in early age was found for hepatocytes isolated from male WAG/Rij and female BN/Bi rats, suggesting that the decline in protein synthesis is sex and strain dependent. In contrast, the increase in protein synthesis in advanced age could be demonstrated with hepatocytes from male WAG/Rij and female BN/Bi rats and therefore seems to be independent of strain and sex. The increase in protein synthesis in advanced age could be attributed to increased excretion of protein in the urine.

Albumin elimination in female WAG/Rij rats with age: a longitudinal study.

A longitudinal study was performed to examine total albumin elimination and urinary albumin excretion in the female WAG/Rij rat. Complete necropsies were performed following the spontaneous death of the animals. The survival characteristics of this group was similar to that of survival cohorts. An increase in total albumin elimination, urinary protein excretion and urinary albumin excretion was observed with age. A proportional increase in the contribution of albumin to the urinary protein excretion was also observed. However, the observed increase in urinary albumin excretion could not totally account for the increase in total albumin elimination. The predominant kidney lesion was chronic progressive nephrosis. The histological severity of the renal lesions were closely correlated with the increase in urinary albumin and total protein loss. It is concluded that the increase in total albumin elimination in rats in this study was due to age-related changes and not to cohort effects.

Changes in fluid-phase endocytosis in the rat with age and their relation to total albumin elimination.

Rates of fluid-phase endocytosis were determined in several organs and tissues of female WAG/Rij rats of several ages by using 125I-labelled polyvinylpyrrolidone ([125I]-PVP) as a marker. Liver, muscle and skin accounted for a high level of [125I]PVP uptake 28 h after injection. When PVP uptake was expressed per gram of organ/tissue, the liver and spleen were found to be the most active. An age-related increase in [125I]PVP uptake was seen at between 12 and 36 months of age in liver, kidneys and heart. Except for the kidneys this increase is caused by an increase in wet weight of these organs and not by an increase in the specific endocytic rate. These data, together with reported findings on the major sites of albumin catabolism, in liver, kidney, spleen and intestine, indicate that fluid-phase endocytosis is a main mechanism for the observed age-related increase in albumin elimination in these rats.

Hepatic cytosolic glutathione S-transferase activities in ageing brown Norway rats--importance of sex differences and phenobarbital treatment for studies of ageing.

Age-associated alterations of hepatic cytosolic glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were investigated in Brown Norway rats of both sexes (11-144 weeks old), under control conditions and after administration of phenobarbital. With both substrates, small changes in glutathione S-transferase activities are observed for the control rats (15-53 weeks old). For these specific age groups, male glutathione S-transferase activities are significantly higher than those of their female counterparts, with sex-related differences being most pronounced after 1,2-dichloro-4-nitrobenzene conjugation. Using 1-chloro-2,4-dinitrobenzene as a 'general' substrate, the sex-differences tend to decrease from the age of 53 weeks onwards to become non-significant at the age of 112 weeks. Phenobarbital administration significantly increases the total and the isoenzymes 3-3 and 3-4 activities in both sexes, with the highest and the lowest increase being observed in the youngest and oldest animals, respectively. It therefore can be concluded that some age-related variations exist as far as the glutathione S-transferase activity of both control and phenobarbital-treated rats are concerned, but that the changes observed are rather small. On the contrary, the parameters 'Sex' and 'Phenobarbital treatment' are found to be responsible for the major activity changes observed.

The relationship between phenazone (antipyrine) metabolite formation and theophylline metabolism in healthy and frail elderly women.

The influence of aging on the metabolism of phenazone (antipyrine), and the relationship between the formation of 3 phenazone metabolites and the metabolic clearance of theophylline in healthy and frail elderly women, were examined. Whereas the elimination half-life did not change, clearance of phenazone decreased by about 50% with age in healthy women receiving phenazone without theophylline. However, the summation of the urinary recovery of phenazone and the measured metabolites, expressed as percentage of the phenazone dose, was lower in the healthy elderly (37 +/- 9% vs 74 +/- 15%). In both healthy and frail females the clearance of formation of 4-hydroxy-phenazone and the metabolic clearance of theophylline correlated strongly (r = 0.93 and 0.90, respectively). In non-healthy elderly females, strong correlations were also observed between the other metabolic pathways of phenazone and the metabolic clearance of theophylline. Coadministration of theophylline in the elderly increased the percentage of the phenazone dose excreted as the measured metabolites. A considerably higher interindividual variability in the disposition of phenazone and theophylline was observed in the frail elderly women. This high degree of variability in drug metabolism may be one of the explanations for the problems often occurring after drug prescription in the elderly.

Pharmacokinetic-EEG effect relationship of midazolam in aging BN/BiRij rats.

1. The purpose of the present investigations was to determine the influence of increasing age on the pharmacokinetics and pharmacodynamics of midazolam in male BN/BiRij rats as an animal model of aging. 2. Midazolam was administered intravenously at a dose of 2.5 mg kg-1 and its pharmacokinetics were determined on the basis of plasma concentrations as measured by high performance liquid chromatography (h.p.l.c.). Pharmacodynamics were studied using the midazolam-induced changes in the electro-encephalogram (EEG) as a measure of the pharmacological effect. Results were evaluated on the basis of simultaneous pharmacokinetic-pharmacodynamic modelling. In an attempt to differentiate between the effects of aging and of concurrent disease, an extensive clinical biochemical/pathological examination was conducted in individual rats by an independent pathologist. 3. The pharmacokinetics of midazolam were best characterized on the basis of a two exponential model. In the 4-month-old rats the values of the clearance, volume of distribution and elimination half-life were 104 +/- 13 ml min-1 kg-1 (mean +/- s.e. mean), 3.4 +/- 0.7 l kg-1 and 30 +/- 3 min, respectively. With increasing age, no changes in the pharmacokinetics of midazolam were observed. 4. The pharmacodynamics of midazolam were determined on the basis of the sigmoidal Emax model. In the 4-month-old rats the values of the parameters relative maximum effect, midazolam concentration at half maximum effect and Hill factor were 106 +/- 10%, 50 +/- 6 micrograms l-1 and 1.6 +/- 0.3, respectively. In the group as a whole no significant changes in the pharmacodynamic parameters of midazolam were observed.4. The pharmacodynamics of midazolam were determined on the basis of the sigmoidal Emax model. In the 4-month-old rats the values of the parameters relative maximum effect, midazolam concentration at half maximum effect and Hill factor were 106 +/- 10%, 50 +/-6 lg 1' and 1.6 +/- 0.3, respectively. In the group as a whole no significant changes in the pharmacodynamic parameters of midazolam were observed. However, when diseased animals were excluded from the evaluation, a tendency towards a decrease in the midazolam concentration at half maximum effect to 25 +/- 14 pg 1-1 was observed in the 36-month-old rats.5. These findings suggest, that increasing age is associated with a tendency towards an increased brain sensitivity to midazolam, which is reflected in a parallel shift of the concentration vs. EEG effect relationship towards lower concentrations. However, it appears that factors other than age also contribute to interindividual variability in pharmacodynamics, considering the substantial interindividual variability within certain age groups.

Pharmacodynamics of the anticonvulsant effect of oxazepam in aging BN/BiRij rats.

1. The purpose of this investigation was to examine the influence of increasing age on the pharmacokinetics and the time course of the anticonvulsant response of oxazepam in BN/BiRij rats as an animal model of aging. 2. Oxazepam was administered intravenously in a dose of 12 mg kg-1 body weight and the anticonvulsant effect intensity was measured as elevation above baseline of a threshold for induction of localized seizure activity (TLS). Direct cortical stimulation with ramp shaped electrical pulse trains of increasing intensity was used to determine this threshold. 3. The pharmacological effect vs. time profile showed in young rats an anticonvulsant component followed by proconvulsant component which is suggestive for the occurrence of acute tolerance and/or withdrawal syndrome. With increasing age the proconvulsant component disappeared, resulting in a monophasic effect profile (anticonvulsant effect only) at the age of 35 months with significantly higher anticonvulsant effect intensity immediately following drug administration. No age-related changes in the pharmacokinetic parameters of oxazepam were observed. 4. In five animals of each age group, benzodiazepine receptor binding characteristics were determined in vitro with [3H]-flunitrazepam as a ligand. Both receptor density and affinity did not show age-related changes. Available literature data on post-receptor events do not indicate conclusive age-related changes. 5. It is concluded, that the observed change in the pharmacodynamics of anticonvulsant effect of oxazepam can be explained by the disappearance of the tolerance/withdrawal phenomenon. This is compatible with a decreased efficiency of homeostatic control mechanisms in the elderly.

Functional properties of rat liver protein synthesizing machinery in relation to aging.

It is well established (van Bezooijen et al., 1977a) that the protein synthesis by isolated liver parenchymal cells (ILPC) from Wag/Rij rats is dependent in a characteristic way on the age of the donor. Whereas cells of middle-aged animals exhibit a decrease in proteo-synthesis activity under in vitro incubation conditions as compared to cells of young rats, a marked increase is observed between 24 and 36 months of age. To investigate whether this overall age-related variation is directly correlated with an intrinsic change at the level of the protein synthesis apparatus of hepatic cells, we compared the size distribution and the in vitro translational activity of polysomes from four age groups. We show that both exhibit the same biphasic response to aging as does the protein synthesizing capacity of ILPC.

Effect of different doses of 3-methylcholanthrene on the localization of the 3-methylcholanthrene-inducible isoenzymes of cytochrome P450 within the centrilobular and periportal zones of the rat liver.

Immunohistochemical staining techniques were used to investigate the localization of the 3-methylcholanthrene inducible isoenzymes (P450 IA1 and IA2) in the rat liver. The rats were induced with different doses of 3-methylcholanthrene, ranging from 2.5 to 25 mg/kg body weight. A heterogeneous induction pattern was observed with induction doses of 2.5; 5; 7.5 and 10 mg/kg body weight with the highest concentration of the isoenzymes around the central vein. With a dose of 25 mg/kg body weight, a homogeneous pattern was found. Induction with a dose of 15 mg/kg body weight resulted in an intermediate situation.

The influence of ageing on the induction of the mRNAs of rat liver cytochromes P450IIB1 and P450IIB2.

To investigate the influence of age on the regulation of the cytochromes P450IIB1 and P450IIB2 the levels of the messenger RNAs for these two cytochromes were determined in liver cytoplasmic RNA of rats of various ages after maximal induction with either phenobarbital or isosafrole and in untreated rats. The levels of these mRNAs were determined by solution hybridization with a RNA-probe (riboprobe system) complementary to both mRNAs. This study showed a marked decrease in the maximal induction levels of these mRNAs between the ages of 12 and 36 months irrespective of the type of inducer used. To assess whether this age-related decrease could be found for both individual mRNAs also solution hybridization experiments were performed with deoxyoligonucleotide probes of a defined sequence. The data presented in this paper show that ageing influences the levels of both the cytochrome P450IIB1 and P450IIB2 mRNA in a similar way. After induction the amount of mRNA for P450IIB1 was in all age groups measured four- to five-fold higher than that of P450IIB2. These data indicate that previously observed age-related changes in the cytochrome P450 system could be related to a lower accumulation of its mRNAs.

Effect of the aging process on the gender and phenobarbital dependent expression of glutathione S-transferase subunits in brown Norway rat liver.

The effect of age, gender and phenobarbital treatment on the hepatic cytosolic glutathione S-transferase subunit composition was studied in Brown Norway rats. Affinity chromatography followed by reversed phase HPLC was used in order to separate the various glutathione S-transferase subunits. Corresponding steady-state mRNA levels were measured by Northern Blot analysis using cDNA clones hybridizing to mRNA encoding glutathione S-transferase subunits 1/2, 3/4 and 7, respectively. In all the age groups studied (15, 25, 53, 99, 112 and 136 weeks) the total amount of glutathione S-transferase protein was in untreated rats significantly higher in males (132 micrograms/mg cytosolic protein) than in females (91 micrograms/mg cytosolic protein) and significant gender dependent differences in the subunit composition were demonstrated. Aging seemed to be of minor importance in untreated as well as in phenobarbital treated rats. Under control conditions, the subunit composition of male rats between 15 and 136 weeks old consisted of 28, 12, 11 and 49% of subunits 1, 2, 3 and 4 respectively and of female animals of the same age groups of 38, 26, 7 and 30%, respectively. In all the age groups studied phenobarbital administration (45 mg/kg body weight, i.p., once a day for 7 days) doubled total glutathione S-transferase protein in both genders and affected the subunit composition in a significant way, emphasizing the already existing differences between genders. Subunits 1, 2 and 3, especially, were increased in male rats in comparison to females resulting in the observation that levels of glutathione S-transferase subunits studied became higher in males than in their female counterparts. The HPLC results were confirmed by steady-state mRNA analysis. In untreated rats, higher levels of mRNA encoding glutathione S-transferase subunits 1/2 and 3/4 were present in male than in female livers. Phenobarbital treatment increased mRNA levels in both genders. Subunit 7 was never detected. These effects were demonstrated in both young and old rats.

Molecular aspects of age-related changes in albumin synthesis in female WAG/Rij rats.

The rate of albumin gene transcription and the level of albumin RNA sequences in liver nuclei of rats of various ages were determined to investigate the regulation of age-related changes in levels of cytoplasmic albumin mRNA in rat liver. No change was observed with age in both the rate of albumin gene transcription and the amount of nuclear albumin RNA sequences, which suggests that the increase in cytoplasmic albumin mRNA content in the rat liver with age is caused by a decreased turnover of this messenger. The length of poly(A)-tails of rat liver cytoplasmic RNA with age was also examined. Although no change with age was observed in the poly(A)-tail length sedimenting at the peak fraction, a shift towards shorter poly(A)-tails was found with age in the overall poly(A)-tail length distribution.