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The mechanism of cadmium-mediated acute hepatotoxicity has been the subject of numerous investigations and although some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process. Acute hepatotoxicity involves two pathways, one for the initial injury produced by direct effects of cadmium and the other for the subsequent injury produced by inflammation. Primary injury appears to be caused by the binding of Cd2+ to sulfhydryl groups on critical molecules in mitochondria. Thiol group inactivation causes oxidative stress, the mitochondrial permeability transition, and mitochondrial dysfunction. Although cadmium may injure hepatocytes directly, there are compelling reasons to believe that hepatocellular injury is produced in vivo as the result of ischemia caused by damage to endothelial cells. Secondary injury from acute cadmium exposure is thought to occur from the activation of Kupffer cells and a cascade of events involving several types of liver cells and a large number of inflammatory and cytotoxic mediators. In this regard, it is clear that Kupffer cell activation and neutrophil infiltration are important events in the toxic process, and the involvement of proinflammatory cytokines and chemokines has also been implicated. The precise roles of the soluble mediators of inflammation warrant further investigation.

In the previous study we showed that senescent male Fischer 344 rats were resistant to Cd-induced hepatotoxicity compared with young-adult rats. In the present study we investigated the role of Kupffer cells and inflammatory cytokines in this effect of aging. The phagocytic activity of Kupffer cells, determined as the removal of carbon from blood, was stimulated by the administration of a hepatotoxic dose of Cd (3 mg/kg sc) in young-adult (5 months) rats but not in old (28 months) rats. Hepatic concentrations of interleukin (IL)-1beta and cytokine-induced neutrophil chemoattractant (CINC), but not of tumor necrosis factor-alpha or IL-6, were elevated in young rats treated with Cd. In old rats, however, the increase in IL-1beta produced by Cd was not statistically significant and the increase in CINC was much lower than in young-adult rats. Pretreatment with gadolinium chloride or cyclosporin A inhibited the elevations in hepatic cytokines and attenuated Cd-induced liver damage, assessed on the basis of serum alanine aminotransferase and sorbitol dehydrogenase activities. Cd-induced hepatotoxicity in the different treatment groups correlated well with hepatic levels of CINC (r = 0.98, p < 0.001) but not with those of IL-1beta. The results suggest that (1) Kupffer cell activation is essential for inflammatory liver damage from Cd, (2) IL-1beta and CINC are important mediators of the inflammatory response induced by Cd, and (3) the attenuation of Cd-induced liver injury in senescent rats is caused by an impairment in Kupffer cell activation, leading to a lower production of CINC and less inflammatory liver injury., (Copyright 2000 Academic Press.)

The influence of aging on the sensitivity of the liver to the acute toxicity of cadmium has not been studied previously in adult rats. In this study hepatotoxicity caused by a single sc injection of CdCl(2) was compared in 5-, 18-, and 28-month-old male Fischer 344 rats. Doses of Cd were adjusted on the basis of the mean lean body mass for each age group of rats, and liver injury was evaluated 24 h after treatment. Cd treatment produced substantial increases in serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 5- and 18-month-old rats, whereas no significant increases were observed in 28-month-old rats. Histologic examination of representative livers from each age group confirmed the findings for serum enzyme activity; hepatocellular necrosis was observed only in livers from 5- and 18-month-old rats. The attenuation of Cd hepatotoxicity in senescent rats did not appear to be related to pretreatment levels of metallothionein or glutathione. Likewise, resistance to Cd could not be explained on the basis of metallothionein induction, which decreased as a function of aging. Thus, the mechanisms that account for the postmaturational decline in sensitivity to Cd do not appear to be associated with alterations in levels of the major factors that protect against Cd-induced hepatotoxicity., (Copyright 1999 Academic Press.)

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.

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.

The purpose of this study was to determine whether the major thiol-disulfide oxidoreductase activities of the rat liver were altered as a consequence of aging, and whether the alterations had any consequences in terms of hepatic thiol concentrations. Liver fractions were prepared from male and female Fischer 344 rats at ages representing young adulthood (5 months), middle age (15 months) and old age (24-29 months), and the activities of the major thiol-disulfide exchange enzymes, together with protein and nonprotein sulfhydryl contents, were measured using spectrophotometric procedures. Thioltransferase, protein disulfide isomerase and thioredoxin reductase activities in livers of male and female rats were unchanged with aging, while glutathione disulfide (GSSG) reductase activity remained the same (in male livers) or increased (in female livers) as a consequence of aging. Both protein and nonprotein sulfhydryl concentrations were well maintained in old age. The absence of age-dependent alterations in the thiol-protein disulfide exchange enzymes and the lack of compromise in the glutathione GSSG reductase system suggest that aged livers retain their capacity to regulate their thiol-disulfide redox balance under normal physiological conditions.

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.

Limited information is available regarding age-associated events that lead to differences in vulnerability to chemicals that injure the liver. For some agents, such as allyl alcohol, alterations in metabolic activation, by liver biotransformation enzymes, are responsible for age-associated changes in severity of liver damage. For other toxicants, such as carbon tetrachloride, there appears to be no relation between changes in activation/detoxification processes and the effects of aging on the extent of liver injury. With diquat, a rise in iron content seems to explain the increased toxicity observed in hepatocytes of old rats compared with those of young-adult rats. Additional research is needed to identify the mechanisms responsible for age-dependent differences in sensitivity to environmental chemicals.

Our previous studies in rat hepatocytes demonstrated an age-dependent increase in sensitivity to diquat-induced cytotoxicity, possibly as a result of increased iron availability. The present study was conducted to determine whether quantitative or qualitative changes in hepatic ferritin occur as a consequence of aging and whether diquat-mediated oxidation is intensified by elevated ferritin concentrations. Hepatic ferritins were isolated from male Fischer 344 rats ages 5, 15, and 25 months. Age-associated increases were observed in amounts of ferritin protein and ferritin iron per gram of liver, but there were no differences in proportions of H to L subunits or in rates of diquat-mediated iron release. The consequences of a threefold increase in ferritin content for diquat-mediated lipid peroxidation and protein carbonyl formation were examined in microsomal incubation systems. The addition of isolated rat liver ferritin augmented diquat-mediated oxidative damage in a time- and concentration-dependent manner, and the inclusion of deferoxamine completely inhibited the stimulation by ferritin. The results indicate that availability of ferritin iron is an important determinant of diquat-mediated oxidative injury and support the hypothesis that elevated hepatic ferritin content is responsible, at least in part, for the age-associated enhancement of diquat-induced toxicity.

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.

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.

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.

The role of altered energy homeostasis in the lethality of allyl alcohol to isolated rat hepatocytes was studied. ATP, ADP, AMP, and viability loss (leakage of lactate dehydrogenase into the medium) were measured in isolated hepatocytes of fed or fasted rats exposed to 0.5 mM allyl alcohol. Adenine mononucleotides and cytotoxicity were determined also in hepatocytes incubated with allyl alcohol in the presence of 4 mM sodium azide or 15 mM fructose. Allyl alcohol-induced cell death in hepatocytes of fed rats was preceded by slight decreases in ATP content and energy charge (16% and 12%, respectively). More substantial decreases in these parameters occurred in parallel with cell killing, but the effect of allyl alcohol on energy status did not exceed the effect produced by a nonlethal concentration of sodium azide. Neither azide nor fructose affected the development of allyl alcohol cytotoxicity. Moreover, allyl alcohol-induced cytotoxicity was similar in hepatocytes of fed and fasted rats. The results suggest that altered energy homeostasis is a consequence rather than a cause of allyl alcohol-induced hepatocyte lethality.

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.

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.

We examined the effect of a toxic concentration of allyl alcohol (0.5 mM) on intracellular calcium concentrations in isolated rat hepatocytes. An increase in phosphorylase a activity was evident in the hepatocytes after 30 min of incubation with allyl alcohol, suggesting that the toxicant may produce an early rise in cytosolic free calcium. The increase in phosphorylase a activity was not reversed by the addition of dithiothreitol (DTT), a sulfhydryl compound that reverses the events that initiate cell killing by allyl alcohol. When intracellular calcium concentrations were measured directly, using fura-2 as the calcium indicator, there was no effect of allyl alcohol on cytosolic free calcium during the first 60 min of exposure, a critical period for development of irreversible damage. Incubation with allyl alcohol did not interfere with the measurement of intracellular calcium. The increases in cytosolic free calcium produced by phenylephrine or ATP were similar to those reported by others and not affected by the presence of allyl alcohol. The results from this study demonstrate that increased cytosolic free calcium is not essential for allyl alcohol-induced cytotoxicity to isolated rat hepatocytes.

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.

The ability of concurrent intraperitoneal injections of endotoxin (0.1 micrograms/kg) and galactosamine (700 mg/kg) to produce liver damage was determined in fasted C57Bl/6 mice of different ages: 2 months (young), 6 months (mature), and 24 months (aged). Liver damage was assessed after 6 hr by measurement of plasma alanine aminotransferase activity (ALAT, mumole/liter/min) and by histological examination for mature and aged mice. Control mice, those treated with saline, galactosamine, endotoxin, or hydrazine alone, had ALAT activities which ranged from 13 to 72 (n = 21). Plasma ALAT activities were increased to hepatotoxic values in some, but not all, mice injected with both endotoxin and galactosamine. For young mice, 7/11 had increased plasma ALAT activities; for mature mice, 5/8 had increased plasma ALAT activities and substantial centrilobular necrosis, whereas for aged mice, 0/7 had increased ALAT activities and none had centrilobular necrosis. Basophilic staining of the cytoplasm was increased by administration of endotoxin and/or galactosamine in both mature and aged mice whether or not necrosis was present. A 5-hr pretreatment with hydrazine sulfate (80 mg/kg) substantially decreased the ALAT release caused by endotoxin and galactosamine in mature mice. Hydrazine pretreatment prevented centrilobular necrosis in mature mice and decreased basophilic cytoplasmic staining in aged mice. The results demonstrate that aged mice are resistant to the hepatotoxic effects of endotoxin and galactosamine which were observed in both young mice and mature mice. Also, hydrazine sulfate pretreatment will protect against the hepatotoxic effects as well as the lethal actions of endotoxin and galactosamine.

The aim of this study was to determine the influence of aging on diquat-induced redox cycling in liver microsomes and diquat hepatotoxicity in rats. Diquat-stimulated production of superoxide anion radical and NADPH-cytochrome c (P-450) reductase activity were measured in liver microsomes prepared from male Fischer 344 rats at ages representing young adulthood (5-6 months), middle age (15-16 months), and old age (24-27 months). Both activities were decreased substantially (40%) in old rats. Diquat-induced liver damage was assessed 6 hr after the administration of diquat (0.1 mmol/kg, ip) on the basis of serum ALT and sorbitol dehydrogenase activities, hepatic microsomal cytochrome P-450 loss, and histological evaluation. The classical manifestations of hepatotoxicity in diquat-treated rats were as severe in old rats as in young-adult ones, despite the age-associated drop in redox cycling capacity. Diquat treatment also resulted in decreased concentrations of hepatic glutathione and ascorbic acid, increased concentrations of hepatic nonheme iron, and decreased liver weights. The changes in glutathione, nonheme iron, and liver weight were more pronounced in livers of middle-aged and old rats than in those of young-adult rats. These age-dependent differences could not be explained on the basis of plasma diquat concentrations, which were similar in the three age groups of rats. The absence of an effect of aging on the hepatotoxic effects of diquat indicates that redox cycling capacity is not limiting for the development of liver damage. Other effects of diquat were influenced by aging, but their relevancy to the hepatotoxicity is uncertain.

The effects of postmaturational aging on the toxicity of diquat, a redox cycling compound, were investigated in hepatocytes that were isolated from mature (6 months) and old (27 months) male Fischer 344 rats and pretreated with 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU), an inhibitor of glutathione reductase. The hepatocytes were incubated for 2 hr with 0, 0.5, or 2.0 mM diquat dibromide, and samples were taken at various time points for measurements of glutathione, glutathione disulfide, thiobarbituric acid reactive substances, lactate dehydrogenase leakage, protein sulfhydryl groups, and protein carbonyl groups. Diquat cytotoxicity was intensified in hepatocytes of old rats compared with those of mature rats, and the enhanced toxicity was associated with increased lipid peroxidation and protein carbonyl formation. However, the enhanced toxicity in old rat hepatocytes was also accompanied by a decrease in diquat-induced GSH oxidation and there was no difference in protein sulfhydryl loss. Concentrations of total nonheme iron and low-molecular-weight chelatable Fe2+, measured with ferene as the chromogen, were several times higher in freshly isolated hepatocytes of old rats than in those of mature rats. We hypothesize that the age-associated enhancement of diquat toxicity could be due to an increased availability of iron for reaction with diquat-generated hydrogen peroxide and for stimulation of lipid and protein oxidation.

The effect of age on the toxicity of diquat, a redox cycling compound, was investigated in hepatocytes isolated from mature (6 months) and old (24-29 months) male Fischer 344 rats. Hepatocytes of old rats were more sensitive than those of mature rats to diquat-induced cytotoxicity (lactate dehydrogenase release into the medium). Cell death was preceded by glutathione disappearance, and rates of glutathione depletion were similar in mature and old hepatocytes. In contrast, diquat-induced formation of thiobarbituric acid-reactive substances was much greater in the hepatocytes from old rats, suggesting that increased lipid peroxidation caused the enhanced cytotoxicity. Further experiments revealed that: 1) hepatocytes of mature and old rats were equally sensitive to iron-induced lipid peroxidation; 2) diquat-stimulated production of superoxide anion radical in liver microsomes did not increase with age, but decreased 43%; 3) superoxide dismutase activity was similar in hepatocytes of mature and old rats; 4) inhibition of catalase activity (which diminishes with age in male rats) did not increase diquat toxicity; and 5) malondialdehyde disappearance in intact hepatocytes decreased (33%) with age, but the toxicological significance of the decline in metabolism was uncertain. Thus, the results demonstrated that diquat-induced lipid peroxidation and cytotoxicity increase with age in male rat hepatocytes, but the enhanced sensitivity to diquat poisoning remains unexplained.

Antioxidant defenses within liver mitochondria are pivotal in preventing liver damage from oxidative toxicants. In this study we determined the activities of glutathione peroxidase (GPO), superoxide dismutase (SOD) and glutathione reductase (GRD) in mitochondria from livers of variously aged Fischer 344 rats. A mixed pattern of age-associated alterations in mitochondrial antioxidant activities was observed. In male rats, GRD activity decreased in old age, whereas GPO and SOD activities increased. In female rats, GPO activity decreased with age, but SOD activity increased and GRD activity was unchanged. Age-associated decreases in antioxidant protection from mitochondrial enzymes appeared to be counterbalanced by increases in protection from other enzymes.

The purpose of this study was to determine the effects of aging on the activities of rat liver enzymes involved in the synthesis and degradation of glutathione, as well as those important for maintaining glutathione in its reduced form. gamma-Glutamylcysteine synthetase, gamma-glutamyltransferase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were measured in liver fractions prepared from male and female Fischer-344 rats at the ages 4, 14, and 29 months. The activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and overall rates of glutathione synthesis were unaffected by aging. In contrast, the activity of gamma-glutamyltransferase, the key enzyme in glutathione degradation, was markedly influenced by animal age; activity increased with age in male rats and decreased with age in female rats. Age-associated alterations also were observed in activities of hepatic enzymes needed to maintain glutathione in its reduced form. Glutathione reductase activity was greater in old female rats and glucose-6-phosphate dehydrogenase activity was greater in old male rats than in young and middle-aged rats of the same sex. Age-dependent changes in glutathione metabolizing enzymes could have important toxicological implications.

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.

The effect of chronic ethanol administration on pulmonary antioxidant protection systems was investigated in male Sprague-Dawley rats exposed to room air or room air containing ethanol vapors for 5 weeks. Blood ethanol concentrations in ethanol-exposed rats were usually between 200 and 300 mg/dl. Glutathione, vitamin E, and malondialdehyde concentrations were measured in lung homogenates, and antioxidant enzyme activities (catalase, glutathione peroxidase, Cu/Zn-superoxide dismutase, glutathione reductase) were determined in the supernatant fractions. For comparison, the measurements were also made using liver fractions. Ethanol treatment increased the activities of catalase (117%) and Cu/Zn-superoxide dismutase (25%) in lung but not in liver. Although chronic ethanol inhalation lowered hepatic glutathione (19%) and hepatic vitamin E (33%), there was no increase in malondialdehyde content in either liver or lung of ethanol-exposed rats. The elevation of pulmonary antioxidant enzyme activities could be interpreted to mean that lung is a target for ethanol-induced oxidative stress. However, as there was no loss of pulmonary GSH or vitamin E and no increase in malondialdehyde formation, it appears that long-term ethanol exposure did not produce a significant degree of oxidative stress in rat lung.

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.

Rates of ethanol metabolism by alcohol dehydrogenase, the microsomal ethanol oxidizing system (MEOS), and catalase were similar in liver preparations from young (4-5 months) and old (24-27 months) female Fischer 344 rats. On the other hand, rates of acetaldehyde metabolism by mitochondrial aldehyde dehydrogenase (ALDH) were 15-20% lower in livers of old rats than in those of younger ones. Results with the ALDH inhibitor cyanamide indicated that a decline in ALDH activity of this magnitude would not increase acute ethanol hepatotoxicity.

The effect of dietary vitamin C on the reduction of cytochrome c and cytochrome P-450 by NADPH-cytochrome P-450 reductase was determined in guinea pig liver microsomes. Acute vitamin C deficiency decreased hepatic microsomal cytochrome P-450 content 21% and the rate of cytochrome P-450 reduction 66% without affecting cytochrome c reduction. However, the vitamin status of the animals did not affect the enhancement in cytochrome P-450 reduction produced by the addition of a type I substrate to the reaction mixture. The results suggest that vitamin C deficiency selectivity affects the transfer of electron from NADPH to cytochrome P-450 and that this effect does not result from a change in the spin state of cytochrome P-450.

The influence of aging on the toxicity of allyl alcohol was studied in hepatocytes isolated from male Fischer 344 rats. Initial values for trypan blue uptake, lactate dehydrogenase (LDH) release, alanine aminotransferase release, and glutathione content were similar in cells isolated from rats aged 5, 15, or 26 months. Incubation with 0.1 to 0.8 mM allyl alcohol resulted in a dose- and time-dependent loss of viability. Dose-effect and time-effect curves were displaced to the left with hepatocytes isolated from old rats compared with those from young rats. Intermediate values were found for hepatocytes of middle-aged rats. Inhibition by pyrazole of allyl alcohol-induced LDH release from hepatocytes also was affected by age. Total protection was observed for cells from young rats whereas no protection was found for those of old rats. Cells from middle-aged rats were between the extremes. The results demonstrate that isolated hepatocytes can be used to study age-related differences in hepatotoxicity.

Severity of liver damage 24 hr after i.p. administration of acetaminophen in doses of 0.4 and 0.8 g/kg was evaluated in male Fischer 344 rats at 4, 14 and 25 months of age. Both doses of acetaminophen produced significant elevations of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 4-month-old rats. Enzyme release was somewhat diminished in old age. Hepatic glutathione (GSH) and microsomal cytochrome P-450 concentrations were decreased in rats that received 0.8 g/kg of acetaminophen. The decreases occurred in young-adult and middle-aged rats, but not in old rats. The results demonstrated that old age does not enhance the hepatotoxic effects of acetaminophen in male Fischer 344 rats.

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.

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.

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.

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.

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.

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.

The oxidation of acrolein by aldehyde dehydrogenase was studied in several subcellular fractions of rat liver by measuring acrolein-dependent production of NADH from NAD+. Mitochondrial and cytosolic fractions each contained two aldehyde dehydrogenase activities with Km values for acrolein of 0.4-0.7 mM and 0.015-0.025 mM. Microsomes demonstrated only a high Km (1.5 mM) activity. The low Km activities of mitochondria and cytosol differed in their sensitivity to inhibition by chloral hydrate and in their response to 1 mM MgCl2 (activation vs. inhibition). The metabolism of acrolein by low Km aldehyde dehydrogenase activities was markedly depressed in mitochondrial or cytosolic fractions from rats pretreated with cyanamide (2 mg/kg for 1 hr) or disulfiram (100 mg/kg for 24 hr). The effect of aldehyde dehydrogenase inhibition on allyl alcohol toxicity was determined by pretreating rats with cyanamide or disulfiram prior to treatment with allyl alcohol. Hepatotoxicity was assessed on the basis of elevated serum alanine aminotransferase and sorbitol dehydrogenase activities and the loss of microsomal cytochrome P-450. Pretreatment with the aldehyde dehydrogenase inhibitors enhanced the hepatotoxicity of allyl alcohol in both male and female rats. The results suggest that acrolein metabolism by rat liver aldehyde dehydrogenase isozymes is important for the inactivation of allyl alcohol-derived acrolein.

The activity of alcohol dehydrogenase was measured in liver cytosolic fractions of male Fischer 344 rats at ages representing young adulthood, middle age, and old age. The activities were 1.7 +/- 0.1, 2.3 +/- 0.1, and 2.6 +/- 0.2 mumol/min/g liver in rats aged 4-5, 14-15, and 24-25 months, respectively. Hepatic alcohol dehydrogenase activity in female rats (3.4 +/- 0.2 mumol/min/g liver) was the same in young as in old rats. Castration increased alcohol dehydrogenase activity in young males to levels found in females, and testosterone administration reversed the effect. However, neither physiological nor pharmacological doses of the hormone restored the elevated enzyme activities of old male rats to levels found in young male rats.

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.

The effects on hepatotoxicity of age-associated changes in drug metabolism are not always straightforward. In the case of allyl alcohol hepatotoxicity in male rats, there is a good relationship between increased metabolic activation by liver alcohol dehydrogenase and enhanced hepatotoxicity in old age. With regard to two other hepatotoxicants, some tentative conclusions about the role of metabolism can be drawn, but they must be tempered with caution due to gaps in the available information. Acetaminophen-induced hepatotoxicity is reduced in old age, and decreased formation of the toxic intermediate may be the reason. There is a prominent effect of aging on acetaminophen conjugation, a shift from sulfation to glucuronidation, but this change does not affect total clearance. The situation with carbon tetrachloride is difficult to interpret because the final outcome is unaltered hepatotoxicity in old age. Nevertheless, the available data suggest that an age-associated decrease in activation of carbon tetrachloride is counterbalanced by a loss in resistance to lipid peroxidation. These conclusions are summarized in Table 5. Again, it must be emphasized that all of these age-dependent changes in toxicity could be related to effects on other systems that are not necessarily involved in the metabolism of hepatotoxicants. Future research is needed to identify pathways of metabolic activation and detoxification in which age-dependent changes occur that result in significant changes in hepatotoxicity. The entire sequence of events from changes at the molecular level to their sequelae at the level of the cell, tissue and intact animal should be investigated, and the results should be confirmed in more than one mammalian model of aging. The aim would be to identify basic mechanisms that result in increased hazard for the aged liver from exposure to toxic compounds.

Microsomes were isolated from livers of male Fischer 344 rats at 3 to 5, 14 to 15 and 24 to 25 months of age for the determination of monooxygenase components and drug metabolism activities. Microsomal cytochrome P-450, cytochrome b5, NADPH-cytochrome c reductase activity and phospholipid were decreased in middle-aged and old rats compared with young-adult rats, but the enzymatic reduction of microsomal cytochrome P-450 was unchanged. Drug metabolism activities both decreased and increased as a consequence of aging, depending upon the substrate used. Differences were observed between young-adult and old rats in the CO maximum of reduced microsomal cytochrome P-450, in microsomal fatty acid composition and in the amounts of microsomal polypeptides having molecular weights of 52,500 and 53,000. The substrate selectivity of the age-related alterations in hepatic microsomal drug metabolism may be due to qualitative changes in the cytochrome P-450 and phospholipid components of the monooxygenase system.

Ethanol and imidazole have been shown to cause similar changes in the hepatic drug metabolizing enzymes of rabbit liver. The effects of these agents on hepatic monooxygenase activities were compared in the rat to test whether similar changes might also occur in this species. Ethanol feeding caused 4- to 5-fold increases in rates of the microsomal metabolism of aniline and p-nitrophenol, and smaller increases in rates of dealkylation of 7-ethoxycoumarin and aminopyrine. Ethanol doubled cytochrome P-450 content and increased the staining intensity of a polypeptide with an apparent molecular weight of 54,000 as determined by SDS polyacrylamide gel electrophoresis. Imidazole pretreatment also enhanced rates of the microsomal metabolism of 7-ethoxycoumarin and aminopyrine, but did not affect rates of aniline or p-nitrophenol hydroxylation. In addition, imidazole did not affect cytochrome P-450 content or the electrophoretic profile of microsomal polypeptides. Thus, the effects of ethanol and imidazole pretreatments on hepatic drug metabolism vary substantially between rabbits and rats.

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.