Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei.

A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.

Lipid peroxidation, antioxidant protection and aging.

The free radical hypothesis of aging proposes that deleterious actions of oxygen-derived radicals are responsible for the functional deterioration associated with aging. Because cellular membranes house the production apparatus of these radicals and because membranes suffer great damage from these radicals, modification of membrane lipids has been proposed to play a major role in the process of aging. Although the relationships between lipid peroxidation and aging have been investigated extensively, the studies have produced conflicting results. Increased lipid peroxidation and decreased antioxidant protection frequently occur, but they are not universal features of aging. Instead, age-dependent changes in these parameters appear to be species-, strain-, sex- and tissue specific. Potential correlations between lipid peroxidation and transition metal concentrations or between lipid peroxidation and declining antioxidant protection have been obscured by the contradictory nature of the findings. Future studies should focus on new approaches for the measurement in vivo lipid peroxidation and on identification of the critical targets of lipid peroxidation.

Effect of aging on aqueous-phase antioxidants in tissues of male Fischer rats.

The purpose of this study was to determine the influence of aging on concentrations of the important aqueous-phase antioxidants in rat tissues. Ascorbic acid, glutathione and uric acid were measured in tissues and organs of male Fischer 344 rats at 6, 15 and 26 months of age. Blood, liver, lungs, heart, kidneys, brain, testes and lenses were excised rapidly and were extracted with cold metaphosphoric acid. Aging diminished the concentration of ascorbic acid in liver, lung and lens; levels in 26-month-old rats were 40-60% of those in 6-month-old rats. Glutathione content was diminished only in lens, where it decreased almost 50% between 15 and 26 months. Some age-associated increases in antioxidant levels also were seen; testis ascorbic acid and kidney glutathione levels were elevated in the old compared with the younger rats. Uric acid concentrations were much lower than glutathione or ascorbic acid concentrations in every tissue except plasma. Old rats had lower levels of uric acid in liver but higher levels in heart, kidney and testis. These results demonstrate that aqueous-phase antioxidant levels are not uniformly diminished in tissues of old rats.

Sex-dependent differences in the effects of aging on antioxidant defense mechanisms of rat liver.

Information about age-related factors that influence sensitivity to hepatotoxic injury is important to geriatric medicine and environmental health. The purpose of the present study was to determine whether age-associated changes occur in hepatic antioxidant defense mechanisms of male and female Fischer 344 rats. Liver homogenates and post-mitochondrial supernatant fractions from rats aged 4, 14, 24 and 29 months were analyzed for antioxidant enzyme activities and for vitamin E and malondialdehyde content. Age-associated changes in catalase and glutathione reductase activities were observed that could be described as sex-determined differences that disappeared in old age. Cytosolic superoxide dismutase and glutathione peroxidase activities displayed sex-dependent variations in activity but were unaffected by aging. Hepatic vitamin E concentrations were lower in male rats than in female malondialdehyde concentrations also were lower in males than in females; malondialdehyde content increased in old males and decreased in old females. The results indicate that age-associated changes in enzymatic and nonenzymatic antioxidant defense mechanisms of rat liver are sex-dependent. In addition, comparison with findings from other studies in rats suggests that the effects of aging may also depend on the strain of rat.

Effects of ethanol on microsomal drug metabolism in aging female rats. I. Induction.

The purpose of this study was to determine how aging affects the induction by ethanol or acetone of the hepatic microsomal monooxygenase system of female Fischer 344 rats. Young-adult, middle-aged and old rats (4, 14 and 25 months) were fed an ethanol-containing or control liquid diet for 15 days. Cytochrome P-450, cytochrome c reductase, aniline hydroxylase, nitrophenol hydroxylase, nitroanisole O-demethylase and benzphetamine N-demethylase activities were measured in hepatic microsomes. All of the drug metabolism activities except benzphetamine N-demethylase were 20-35% lower in old than in young-adult rats fed the control diet. In addition, the increase in drug metabolism produced by feeding the regular ethanol diet (36% of calories as ethanol) was 50-60% lower in the old rats. However, there was no difference in the magnitude of ethanol induction when ethanol intakes were matched. The effects of chronic acetone consumption (1.2g/day per kg body weight for 15 days) paralleled those of ethanol consumption, except that the extent of induction was greater with acetone. Acetone-induced levels of hepatic microsomal cytochrome P-450, nitrophenol hydroxylase, nitroanisole O-demethylase and aniline hydroxylase were similar in all three age groups. The results of this study indicate that induction of hepatic microsomal drug metabolism by ethanol or acetone is unaffected by the aging process.

Effects of ethanol on microsomal drug metabolism in aging female rats. II. Inhibition.

The effect of aging on the inhibition by ethanol of drug metabolism activity was examined in liver microsomes of female Fischer 344 rats aged 4, 14 and 24 months. Inhibition of aniline hydroxylase activity in microsomes from 4-month-old females occurred at low concentrations of ethanol (0.1 mM) and was predominantly competitive. Aging was associated with a significant increase in apparent Km for aniline in the absence of ethanol (24 +/- 2, 20 +/- 2 and 32 +/- 1 microM in microsomes from 4-, 14- and 24-month-old rats, respectively) and a change from competitive to non-competitive inhibition by ethanol. Inhibition of benzphetamine N-demethylase activity occurred only at high concentrations of ethanol (100 mM) and was non-competitive in nature. There were no significant effects of aging on the kinetics of the reaction or the type of inhibition produced by ethanol. Microsomal ethanol oxidation rates were measured in liver microsomes of 4-, 15- and 25-month-old Fischer 344 rats of both sexes. Ethanol oxidation in males was greater than in females and was decreased significantly in old age. Ethanol oxidation in female rats was unaffected by aging. The results suggest that significant changes in drug/ethanol interactions can occur as a consequence of aging.

Substrate specificity of age-related changes in the inducibility of hepatic microsomal monooxygenases in middle-aged rats.

Hepatic microsomal monooxygenase induction was investigated in young-adult and middle-aged male Fischer 344 rats. Monooxygenase components and drug metabolism activities were determined in liver microsomes prepared from rats treated with phenobarbital (PB, beta-naphthoflavone (BNF) or methyltestosterone (MT) and compared with values from untreated rats. PB and BNF effects on cytochrome P-450 concentration and cytochrome c reductase activity were similar in young-adult and middle-aged animals. However, the extent of cytochrome P-450 induction by MT was less in the older animals. The age-related changes in induction of drug metabolism activities differed with different substrates for the monooxygenase system. In contrast to the inducibility of benzphetamine N-demethylation and aniline hydroxylation, which was diminished in the older rats, the inducibility of nitroanisole O-demethylation was enhanced. The results imply that qualitative changes in the microsomal enzyme system occurred as the animals progress from young to middle adulthood.

Effects of ethanol on microsomal drug metabolism in aging female rats. III. In vivo.

The effects of aging on ethanol inhibition of zoxazolamine metabolism in vitro and in vivo were studied in female Fischer 344 rats aged 4, 14 and 26 months. Zoxazolamine hydroxylase activity in freshly-isolated liver microsomes decreased significantly with age (1.88 +/- 0.32, 1.49 +/- 0.30 and 0.74 +/- 0.18 nmol/min per mg protein in young-adult, middle-aged and old rats, respectively). A substantial inhibition of zoxazolamine hydroxylation occurred in the presence of 40 mM ethanol. The extent of inhibition was the same in microsomes from all three age groups. The effect of aging on the duration of zoxazolamine paralysis in vivo reflected the effect of aging on zoxazolamine metabolism in vitro. Mean duration of paralysis following a standard 50 mg/kg dose of zoxazolamine increased significantly as a function of aging (0.5, 2.9 and 4.7 h in young-adult, middle-aged and old rats, respectively). Administration of ethanol (1.2 g/kg) 10 min before zoxazolamine treatment prolonged the duration of zoxazolamine paralysis in young-adult and middle-aged rats by about 2 to 2.5 h, but ethanol pretreatment did not affect paralysis time in old rats. Thus, the inhibitory effect of ethanol on zoxazolamine metabolism in vivo appeared to be attenuated in old age.

Effect of methyltestosterone administration on microsomal drug metabolism in aging rats.

The effects of methyltestosterone administration (100 mg/kg for four days) on the hepatic microsomal drug-metabolizing system from old male rats was investigated. Age-related decreases in cytochrome P-450 content, cytochrome c reductase activity and benzphetamine N-demethylase activity were unaffected by methyltestosterone treatment. Administration of the androgen induced nitroanisole O-demethylase and aniline hydroxylase activities, resulting in a restoration of the latter to levels found in young-adult animals.

Evaluation by dual-energy X-ray absorptiometry of age-related changes in body composition of male rats.

The purpose of this study was to determine the feasibility of using a recently developed procedure, dual-energy x-ray absorptiometry, to determine body composition in rats as a function of aging. Results were obtained that were consistent with previous findings for male rats.

Carbon tetrachloride hepatotoxicity as a function of age in female Fischer 344 rats.

Severity of liver damage 24 h after intraperitoneal administration of carbon tetrachloride (0.2 ml/kg) was evaluated in female Fischer 344 rats aged 5, 14 and 28 months, i.e. in young adulthood, middle age and old age. Carbon tetrachloride-induced hepatotoxicity, as judged by the leakage of hepatic enzymes into the bloodstream and the disappearance of hepatic microsomal cytochrome P450, was much less severe in old rats than in young-adult rats. For example, serum sorbitol dehydrogenase (SDH) activity following carbon tetrachloride administration was 680 mumol/min/l in old rats compared with 1710 mumol/min/l in young-adult rats, and the loss of hepatic cytochrome P450 was 25% of the total amount in old rats compared with 50% of the total in young-adult rats. Spin trapping and electron spin resonance (ESR) spectroscopy were utilized to measure the conversion of carbon tetrachloride to trichloromethyl radicals in vivo. This primary bioactivation step occurred at similar rates in female rats aged 5, 14 and 28 months. In addition, the total nonheme iron contents in livers of rats in the three age groups were similar. Thus, the age associated attenuation of carbon tetrachloride-induced hepatotoxicity was not explained on the basis of decreased bioactivation to reactive species or decreased availability of iron for promotion of lipid peroxidation. The results suggest that other factors are important determinants of age-associated changes in sensitivity to toxic chemicals.

Age and gender differences in hepatic ascorbic acid concentrations and NADPH-dehydroascorbic acid reductase activity.

Ascorbic acid content decreased with age in livers of male rats but not in those of female rats. The influence of aging on the regeneration of ascorbic acid from dehydroascorbic acid was investigated as a possible cause of declining ascorbate concentrations. The results demonstrate that hepatic NADPH-dehydroascorbic acid reductase activity is unaffected by aging.

Effect of age and carbon tetrachloride on cytokine concentrations in rat liver.

Interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in livers of young-adult and old rats administered carbon tetrachloride or vehicle. IL-1beta levels were higher and IL-6 levels were lower in old rats than in young-adult rats. Carbon tetrachloride treatment increased IL-1beta and decreased TNF-alpha and IL-6. The elevation in IL-1beta was diminished by aging. These results indicate that the increase in carbon tetrachloride hepatotoxicity that occurs in old age could be related to a dysregulation of inflammatory cytokines.

Differential effects of aging on hepatic microsomal monooxygenase induction by phenobarbital and beta-naphthoflavone.

The influence of aging on hepatic microsomal monooxygenase induction by phenobarbital (PB) or beta-naphthoflavone (BNF) was investigated in male Fischer 344 rats maintained in a constant environment. PB-induced increases in microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity were similar in rats aged 3-5 months (young-adult) and 24-25 months (old), but increased in benzephetamine N-demethylase activity were markedly diminished in the old rats. Separation of hepatic microsomal proteins by sodium dodecylsulfate gel electrophoresis demonstrated that aging decreased the induction by PB of a polypeptide with a molecular weight of 52,500. BNF-induced increases in microsomal cytochrome P-450 and nitroanisole O-demethylase activity were greater in old than in young-adult rats, and BNF induction of 55,000 and 57,000 molecular weight microsomal polypeptides was increased slightly in livers from old rats. The results indicate that age-related effects on monooxygenase induction vary with different inducers of the hepatic microsomal enzyme system.

Dithiothreitol reversal of allyl alcohol cytotoxicity in isolated rat hepatocytes.

Reversal by dithiothreitol (DTT) of allyl alcohol cytotoxicity was investigated in isolated rat hepatocytes. Allyl alcohol-induced protein sulfhydryl loss, bleb formation, and cell death were prevented by DTT, when it was added to hepatocytes 30 min after the toxicant. The protective effect of DTT also was demonstrated in cells that were washed after 30 min of exposure to allyl alcohol, indicating that protection was not related to inhibition of allyl alcohol metabolism or inactivation of acrolein. DTT reversed the cell surface protrusions that formed during exposure to allyl alcohol, but reversal of blebbing did not insure that the cells would remain viable. Glutathione disulfide was not formed in allyl alcohol-treated cells, and DTT reversal of cytotoxicity occurred without restoring glutathione levels. Moreover, protection against allyl alcohol toxicity required the continuous presence of DTT. The results suggest that initial events in the toxic process are reversible, and that DTT can prevent cytotoxicity if added to hepatocytes before irreversible damage occurs; however, the mechanism by which DTT exerts its protection is not clear.

Oxidation of pyridine nucleotides is an early event in the lethality of allyl alcohol.

The involvement of altered pyridine nucleotide concentrations in the cytolethality of allyl alcohol was studied in isolated rat hepatocytes. NAD+, NADH, NADP+, NADPH and viability loss (leakage of lactate dehydrogenase into the medium) were measured in cells incubated with 0.5 mM allyl alcohol with or without the addition of 2 mM dithiothreitol at 30 min. Exposure to allyl alcohol increased NADH levels in the first 15 min of incubation. A sharp drop in NADH and NADPH with an accumulation of NADP+ occurred between 30 and 60 min of incubation with allyl alcohol, indicating an oxidation and interconversion of pyridine nucleotides. Dithiothreitol prevented the oxidation of pyridine nucleotides, but not their reduction or interconversion, and protected against cell killing by allyl alcohol. The results suggest that pyridine nucleotide oxidation might be important for allyl alcohol-induced cytotoxicity; however, a causal relationship between pyridine nucleotide oxidation and cell killing is yet to be demonstrated.

Drugs and nutrition in old age.

Drug effects are influenced by physiologic and pathologic changes that occur as a consequence of aging. The elderly may be more disposed to drug-induced nutrient depletion because of chronic illness, inadequate diet and long-term drug use. Digoxin, isoniazid, corticosteroids, diuretics and psychoactive agents pose special hazards to the nutritional status of elderly patients. On the other hand, dietary factors, such as protein levels or vitamin deficiencies, may be important determinants of age-related changes in drug disposition or toxicity.

Effects of acute ethanol administration on female rat liver as a function of aging.

Female Fischer 344 rats, aged 4, 14, and 25 months, received 4.0 g/kg of ethanol by intraperitoneal (i.p.) injection. Blood alcohol concentrations 2.5, 6 and 16 hr after ethanol injection were similar in the three age groups. Hepatic glutathione (GSH) levels were diminished 6 hr after ethanol injection, and there were no age-dependent differences in the depleted levels (3.2 +/- 0.1, 3.5 +/- 0.2, and 3.0 +/- 0.5 micrograms GSH/g liver). However, GSH contents in livers of young-adult rats approached control levels after 16 hr, whereas they remained depressed in older rats. Serum levels of hepatic enzymes were significantly elevated 6 hr after ethanol administration. The increases were greater in middle-aged and old rats than in young-adult rats. The results suggest that middle-aged and old rats are more susceptible than young rats to the acute toxicity of ethanol.

Loss of mitochondrial membrane potential is not essential to hepatocyte killing by allyl alcohol.

Allyl alcohol-induced LDH leakage from isolated rat hepatocytes was preceded by a decrease in rhodmine 123 retention, signifying a loss of mitochondrial membrane potential. Addition of dithiothreitol (DTT) prevented the drop in membrane potential and completely prevented cell killing by allyl alcohol. In contrast, cyclosporin A and trifluoperazine delayed the loss of membrane potential without affecting cytolethality. The results indicate that a drop in mitochondrial membrane potential is not essential for allyl alcohol lethality. The mitochondrial dysfunction produced by allyl alcohol appears to be the consequence of an earlier event in the toxicity that is reversible by DTT.

Serum and liver concentrations of tumor necrosis factor alpha and interleukin-1beta following administration of carbon tetrachloride to male rats.

Inflammatory cytokines are recognized as early mediators of tissue damage and repair. The purpose of this study was to determine the effects of carbon tetrachloride administration on tumor necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) concentrations in serum and liver of rats. Administration of 0.2 ml/kg, i.p., of CCl4 to male Fischer 344 rats caused modest increases in serum levels of both cytokines; elevations of TNF-alpha were statistically significant at 4 and 12 h, and elevations of IL-1beta were statistically significant at 24 h. Although CCl4 produced substantial increases in liver IL-1beta concentrations (more than 3-fold), levels of TNF-alpha were not affected. Treatment with 0.1, 0.32 or 1.0 ml/kg of CCl4 produced dose-dependent increases in serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities, but serum cytokine concentrations were not dose-dependent and did correspond with serum ALT and SDH activities. The results suggest that IL-1beta production in rat liver is stimulated by hepatotoxic doses of CCl4. Production of TNF-alpha may also be induced, but the source of TNF-alpha in serum could be a tissue or organ other than liver.