Microsomal mixed-function oxidase activity and senescence—I. Hexobarbital sleep time and induction of components of the hepatic microsomal enzyme system in rats of different ages

The duration of recovery time of CFN male rats from hexobarbital hypnosis increases with advancing age. Pretreatment of the animals with phenobarbital results in a shortening of recovery time from hexobarbital hypnosis for animals of all ages.The results of in vitro studies of zoxazolamine hydroxylase activity suggest that the age-related differences in hexobarbital recovery time result, in part, from an age-related decrease in the rate of drug metabolism by the hepatic microsomal enzyme system. The decrease in zoxazolamine hydroxylase activity with advancing age is most probably the result of the age-related decrease in NADPH-cytochrome c reductase activity.Treatment with phenobarbital results in the induction of zoxazolamine hydroxylase activity, cytochrome P₄₅₀ content, and NADPH-cytochrome c reductase activity in rodents of all ages. All these functional components of the system are induced to similar levels in adult animals. No age-related lag in the induction of any of these functional components of the hepatic microsomal enzyme system was observed.Although no age-related difference in the functional components of the hepatic microsomal detoxification system were found following phenobarbital treatment, the age-related difference in recovery time from hexobarbital hypnosis following phenobarbital treatment appeared to persist in some individuals.In our judgment, these findings strongly suggest that factors other than the functional state of the hepatic microsomal drug detoxification system, modify the effects of pharmacological agents, such as hexobarbital, on some senescing rodents.     

The effect of age on mono-oxygenase enzyme kinetics in rat liver microsomes

The clearance of many oxidized drugs falls with age. Whilst factors such as reduced liver size, blood flow and specific enzyme activity may be important, the possibility that reduced enzyme affinity for substrate contributes to this fall has not hitherto been investigated. Using liver microsomes from 12 young adult and 12 elderly male Norwegian Brown rats we defined the kinetics of ethoxyresorufin-O-de-ethylation and aldrin epoxidation, specific substrates for the 3-methylcholanthrene inducible and phenobarbitone inducible forms of cytochrome P450, respectively. Our results show a marked fall in the maximal activity of both enzymes in advanced age whether expressed in terms of microsomal protein or unit of cytochrome P450, but with no change in apparent enzyme affinity (Km). Since Km is unchanged, we feel that qualitative age-related changes in cytochrome P450 are unlikely. Reduced metabolism may be due to age-related alterations in coenzymes or smooth endoplasmic reticulum lipid membranes.     

Effects of aging and long-term caloric restriction on hepatic microsomal monooxygenases in female fischer 344 rats: Alterations in basal cytochrome P-450 catalytic activities

The effects of aging and caloric restriction on uninduced levels of hepatic microsomal cytochrome P-450s and specific P-450-related catalytic activities were evaluated in female Fischer 344 rats. Microsomes were isolated from livers of ad libitum (AL) fed rats 1, 3, 6, 16 and 26 months of age, and from calorie-restricted (CR) rats 6, 16 and 26 months of age. The recovery of microsomal protein was higher in CR than AL rats at 6, 16 and 26 months of age. Between ages 3 and 26 months there was a gradual decline in recovered microsomal P-450, but the differences between successive age categories were not significant. A different trend was observed for total cytochrome P-450 per liver. AL rats showed a 2-fold decline in P-450/liver between 6 and 26 months of age, while P-450/liver of corresponding CR rats increased 1.7-fold. Neither cytochrome b₅ nor NADPH cytochrome C reductase varied consistently as a function of increased age, with all age groups showing greater reductase activity for CR rats than for AL rats. Phenobarbital (PB) and methylcholenthrene (MC) inducible cytochrome P-450 catalytic activities (pentoxy [PROD] and ethoxy [EROD] resorufin O-deethylases) exhibited similar age-related alterations that were distinctly different from the two other enzymes examined. Both PROD and EROD showed dramatic declines in activity between 1 and 3 months of age, with a slight increase in activity at 6 months and maintenance of the activity levels through 26 months. Benzphetamine N-demethylase (BND) and aniline p-hydroxylase (APH) activities were also altered as a function of increased age, with CR rats exhibiting increased isozyme activity above that seen for aging AL rats. Calorie restriction appears to have a modulating effect, compensating for an age-related decline of hepatic microsomal monooxygenase activity in female Fischer 344 rats.     

Decline in hepatic microsomal monooxygenase components in middle-aged Fischer 344 rats

Monooxygenase components and drug metabolism activities were determined in liver microsomes from young-adult (3–5 months) and middle-aged (14 months) male Fischer 344 rats. Several components of the monooxygenase system were decreased in middle-aged rats including total cytochrome P-450, cytchtomre b₅, NADPH-cytochrome, reductase activity and phospholipids. However, the reduction of cytochrome P-450 by NADPH, thought to be rate-limiting for monooxygenase activity, was not decreased. Drug metabolism activities in microsomes from middle-aged rats were increased (nitroanisole O-demethylation), decreased (aniline hydroxylation) or unchanged (benzphetamine N-demethylation). Aging decreased the microsomal content of phospholipids and changed the relative percentages of several microsomal fatty acids. The substrate selectivity of the age-related changes in drug metabolism activities may be related to changes in the fatty acid composition of microsomal phospholipids.     

Altered hepatic drug metabolism in senescent mice

Age-related alterations were examined in the system of hepatic drug metabolism in male mice. Although cytochrome P₄₅₀ content and composition did not change, several mixed-function oxidase activities decreased with age. However, both benzo(a)pyrene hydroxylase and 7-ethoxycoumarin de-ethylase showed a significant increase in the senescent mouse, in contrast to the situation previously described in aging rats. Old mice were able to respond to treatment with polychlorinated biphenyls, potent inducers of hepatic drug metabolism. The basis for senescent changes in hepatic drug metabolism still requires elucidation.     

Hepatic microsomal function in rats with chronic dietary iron overload

We determined whether alterations in hepatic microsomal function occur in association with iron-induced lipid peroxidation in vivo in rats with chronic dietary iron overload. In rats fed a 2.0% carbonyl iron diet for a period of 20 wk, there was no significant microsomal conjugated diene formation (evidence of microsomal lipid peroxidation) or difference in cytochrome P450 concentration found at mean (+/- SEM) hepatic iron concentrations of 1210 +/- 92 micrograms/g liver (wet wt) or 2730 +/- 100 micrograms/g. At a hepatic iron concentration of 4090 +/- 245 micrograms/g, however, there was significant conjugated diene formation (p less than 0.001) and a 56% decrease in the cytochrome P450 concentration (p less than 0.001). In rats fed a 2.5% carbonyl iron diet for 10 wk, achieving a liver iron concentration of 4820 +/- 420 micrograms/g, there was significant microsomal conjugated diene formation (p less than 0.001), a 35% reduction in cytochrome P450 (p less than 0.005), and a 16% reduction in aminopyrine demethylase activity (p less than 0.025), but only an 8% reduction in glucose-6-phosphatase activity (p = not significant). Finally, in rats fed a 3.0% iron-supplemented diet for 7 wk, achieving a liver iron concentration of 2730 +/- 205 micrograms/g, there was a 23% reduction in cytochrome P450 (p less than 0.025), a 28% reduction in cytochrome b5 (p less than 0.001), and a 47% increase in heme oxygenase activity (p less than 0.025) (heme oxygenase activity measured in this group only). We conclude that oral iron loading can produce microsomal lipid peroxidation in vivo that is associated with selective decreases in microsomal hemoprotein concentrations and cytochrome P450-dependent enzymes.     

Inhibition of rat methylbenzylnitrosamine metabolism by dietary zinc and zinc in vitro.

Methylbenzylnitrosamine is an esophageal-specific carcinogen in the rat, and the incidence of methylbenzylnitrosamine-induced esophageal carcinoma is increased by dietary zinc deficiency. Methylbenzylnitrosamine requires activation by cytochrome P-450 to be mutagenic; the present study examined the role of dietary zinc deficiency and the in vitro addition of zinc on the cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. Dietary zinc deficiency significantly increased the cytochrome P-450-dependent esophageal and hepatic microsomal metabolism of methylbenzylnitrosamine. These changes occurred without alteration in the specific content of total microsomal cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. These results suggest that physiological levels of zinc may be an endogenous inhibitor of methylbenzylnitrosamine metabolism. Dietary zinc deficiency appears to reduce this inhibition of cytochrome P-450 activity, resulting in an increase in carcinogen activation.     

Studies on age related changes in cytochrome P-450, cytochrome b-5 and mixed function oxidase activity in mouse liver microsomes,in relation to their phospholipid composition

Liver microsomes were isolated from male mice of various defined ages, and their ability to metabolise ethylmorphine, p-nitroanisole, p-aminobenzoic acid and aniline was assessed as was their content of cytochrome P-450, cytochrome b-5 and protein, and their lipid composition.The rising capacity of the microsomes to metabolise these substrates in the first half of the lifespan was associated with a rise in the cytochrome P-450 and cytochrome b-5 content as well as an increase in the ratio of phosphatidylcholine to both phosphatidylethanolamine and sphingomyelin and, in early adult life, decreasing saturation of phospholipid fatty acids. The lipid changes would be expected to increase microsomal membrane fluidity.Declining mixed function oxidase activity in later life paralleled a decline in the ratio of phosphatidylcholine to phosphatidylethanolamine and sphingomyelin, and progressively reduced saturation of phospholipid fatty acids. Cytochrome content showed only a slight irregular decline with advancing age until the oldest animals were investigated, in which cytochrome b-5 was considerably reduced. Unfortunately, cytochrome P-450 was not measured in the oldest available group.Inducing the mixed function oxidase system with daily phenobarbitone injections for 4 days substantially increased enzyme activity in animals up to the middle of the age range tested. Older animals showed progressively reduced induction and none at all was seen in the oldest group. In contrast with the situation in non-induced mice, these changes were more closely associated with changes in cytochrome P-450 content than with alterations of lipid composition.     

Induction of hepatic mixed function oxidases in senescent rodents—II. Effect of polychlorinated biphenyls

Senescent alterations in the ability of the drug-metabolizing system of rat liver to respond to treatment with the potent mono-oxygenase inducer Aroclor 1254, a complex mixture of polychlorinated biphenyls, was analyzed. The contents of hepatic microsomal cytochrome P₄₅₀, NADPH cytochrome c reductase, and lipid were measured as were several coordinated mixed function oxidase activities. The peptide composition was also examined by slab gel electrophoresis. The senescent rodents responded as well or better than younger animals to induction with polychlorinated biphenyls, in certain cases restoring the “young” mono-oxygenase system. Thus, the old rats exhibit no age-related impairment in their response capacity to inducers of hepatic drug metabolism.     

Cytochrome P-450 mediated metabolism in active murine systemic lupus erythematosus

In a comparison of NZB/NZW female mice either with active systemic lupus erythematosus (SLE) (28-33 weeks of age) or without overt SLE (7-13 weeks), the hepatic microsomal activities of ethoxycoumarin O-deethylation and aminopyrine N-demethylation were decreased 32% (p less than 0.05) and 28% (p less than 0.03), respectively, and cytochrome P-450 levels were decreased 34% (p less than 0.01) in the mice with active SLE. These changes were not associated with age differences alone in 2 nonautoimmune strains. Active murine SLE is thus associated with significant depressions in both hepatic cytochrome P-450 levels and microsomal enzyme activities. The metabolism of drugs and endogenous substrates may thus be impaired in active SLE.     

Caloric restriction affects liver microsomal monooxygenases differentially in aging male rats

Caloric restriction (CR) extends life span and retards the onset of physiological changes and pathologies associated with aging, but the underlying mechanisms remain unresolved. This study demonstrates that CR postpones the documented age-related declines in and/or enhances the activity and microsomal concentration of several liver monooxygenases in male rats, i.e., NADPH cytochrome P-450 reductase, total cytochromes P-450. However, the relative concentration of cytochrome P-450b+C did not exhibit statistically significant changes, whereas another isozyme, the male specific P-450h, declined significantly in both ad libitum-fed and CR rats as a function of increasing age. While CR appears to retard age-associated changes in certain liver enzymes, this effect is by no means universal. The hepatic monooxygenases constitute a well-characterized enzyme system in which to examine the perturbation of the aging process by CR.     

Effect of amphotericin B on hepatic cytochrome P-450 and glucose-6-phosphatase in the rat

The effect of amphotericin B on hepatic microsomal cytochrome P-450 (P-450) concentration was measured in vitro, in vivo and ex vivo in the rat. In vitro, both amphotericin B (0–500 μg/ml) and its vehicle, sodium deoxycholate (0–410 μg/ml), caused similar dose-dependent decreases of P-450 concentrations and glucose-6-phosphatase activity. Intravenous amphotericin B (3 mg/kg) given daily for 3 days decreased antipyrine clearance from control values of 1.24 ± 0.24 ml/min to 0.67 ± 0.12 ml/min (p < 0.001); whereas antipyrine clearance was unchanged by sodium deoxycholate. The P-450 concentration on the third day was reduced from 0.74 ± 0.14 nmol/mg protein in control rats to 0.33 ± 0.09 nmol/mg protein in rats treated with amphotericin B (p < 0.001). Sodium deoxycholate had no effect on P-450 concentration. In contrast, amphotericin B had no effect on either antipyrine clearance or P-450 concentration following enzyme induction by phenobarbital. Amphotericin B had no effect on microsomal glucose-6-phosphatase activity in vivo. Neither amphotericin B nor sodium deoxycholate induced lipid peroxidation, measured as malondialdehyde production. These results show that amphotericin B decreases hepatic cytochrome P-450 content and function in the rat. These effects can not be observed in the enzyme induced state. Amphotericin B has no effect on glucose-6-phosphatase in vivo, the key enzyme of the gluconeogenesis, indicating selective effects on hepatic microsomal function.     

Age-related changes in liver drug-metabolizing enzymes

The non-induced levels of hepatic microsomal NADPH cytochrome c reductase and cytochrome P-450 undergo marked age-dependent declines in male Fischer rats, especially between maturity and senescence. Phenobarbital administration causes significant increases in these two parameters in young (1 month) and mature (16 months) animals, but not in senescent rats (27 months). Both the rates of enzyme and hemoprotein induction and the maximum-induced levels achieved after 6 days of drug treatment are significantly greater in the young and mature animals vs the senescent rats. These functional data correlate reasonably well with (1) a similar age-dependent response of microsomal protein concentration to phenobarbital administration and (2) the quantitative morphological evidence demonstrating an age-related loss in the amount of hepatic smooth surfaced endoplasmic reticulum between maturity and senescence.     

Hypoglycemic activity of Hemidesmus indicus R. Br. on streptozotocin-induced diabetic rats

OBJECTIVE:

To evaluate the antidiabetic activity of an aqueous extract of the roots of Hemidesmus indicus on blood glucose, serum electrolytes, serum marker enzymes, liver microsomal P-450 enzymes, and lipid peroxidation in the liver and kidney of streptozotocin-induced diabetic rats.

MATERIALS AND METHODS:

Effect of H. indicus extract on blood glucose was studied with fed, fasted and glucose-loaded diabetic and nondiabetic rat models. The effect of the extract on serum electrolytes, serum levels of key glucose metabolizing enzymes, hepatic microsomal protein and hepatic cytochrome P-450-dependent mono-oxygenase enzyme systems and lipid peroxidation in the liver and kidney of diabetic rats. One way analysis of variance and Duncan''s multiple range test was used for statistical analysis.

RESULTS:

Oral administration of H. indicus aqueous extract to fed, fasted and glucose-loaded diabetic rats decreased blood glucose level significantly at 5 h and restored serum electrolytes, glycolytic enzymes and hepatic cytochrome P-450-dependent enzyme systems by preventing the formation of liver and kidney lipid peroxides at the end of 12 weeks of the study period.

CONCLUSION:

From the studies, it can be concluded that the aqueous extract of the roots of H. indicus at a dosage of 500 mg/kg/day exhibits significant antidiabetic activity. It restores the concentrations of electrolytes, glucose metabolizing enzymes, hepatic microsomal protein and hepatic cytochrome P-450-dependent mono-oxygenase enzyme systems to near normal level and also corrects the related metabolic alterations in experimentally induced diabetic rats. H. indicus administration also decreased liver and kidney lipid peroxidation products. On the basis of our findings, H. indicus could be used as an antidiabetic and antioxidant agent for the prevention and treatment of diabetes mellitus.     

Different effects of short- and long-term dietary choline-deficiency on hepatic microsomal phospholipids and drug oxidation

Prolonged administration of a choline-deficient diet to male rats results in the development of hepatic cirrhosis and alterations in oxidative drug metabolism. The present study was designed to assess whether the changes in drug metabolism were related to the development of cirrhosis or merely to the effects of choline-deficiency on hepatic microsomal lipid composition. Male rats were given a synthetic choline-deficient diet for either 1 week (short-term) or 30 weeks (long-term), and results at each time were compared with age-matched control rats given the same diet but with supplementary choline.
After both 1 week and 30 weeks of the choline-deficient dietary regimen, the proportion of microsomal phospholipid present as phosphatidylcholine was significantly decreased, and that present as phosphatidylethanolamine was significantly increased, compared with appropriate controls. However, microsomal cholesterol content (per mg of microsomal protein) was not significantly changed at either time. Cytochrome P-450 levels and the turnover of ethylmorphine N -demethylase (enzyme activity/nmol cytochrome P-450) were significantly reduced in the cirrhotic (30 week) model whereas short-term intake of the diet did not alter the levels of cither enzyme. These findings suggest that the effects of changes in phosphatidylcholine and phosphatidylethanolamine levels in choline-deficiency cirrhosis have minimal importance with respect to changes in drug oxidation. Instead, altered regulation of specific cytochrome P-450 isozymes appears to be the principal cause of impaired oxidation.     

Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants.

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by cells via induction of nitric oxide synthase. We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimine or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicals that induce distinct molecular isoforms of cytochromes P450 (beta-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe(2+)- and Fe(3+)-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N omega-nitro-L-arginine methyl ester. Our findings implicate NO over-production as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.     

Age-related changes in liver drug-metabolizing enzymes

The non-induced levels of hepatic microsomal NADPH cytochrome c reductase and cytochrome P-450 undergo marked age-dependent declines in male Fischer 344 rats, especially between maturity and senescence. Phenobarbital administration causes significant increases in these two parameters in young and (1 month) and mature (16 months) animals, but not in senescent rats (27 months). Both the rates of enzyme and haemoprotein induction and the maximum-induced levels achieved after six days of drug treatment are significantly greater in the young and mature animals vs the senescent rats. These functional data correlate reasonably well with [1] a similar age-dependent response of microsomal protein concentration to phenobarbital administration and [2] the quantitative morphological evidence demonstrating an age-related loss in the amount of hepatic smooth-surfaced endoplasmic reticulum between maturity and senescence.     

Bile acids produce a generalized reduction of the catalytic activity of cytochromes P450 and other hepatic microsomal enzymes in vitro: Relevance to drug metabolism in experimental cholestasis

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of individual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the disproportionate lowering of male-specific P450 enzymes results from secondary down-regulation of some cytochrome P450 (CYP) genes. We report herein the results of experiments to test the first part of this hypothesis, at least as indicated by enzyme inhibition. Hepatic microsomal fractions from normal male rats were incubated at 37°C with increasing concentrations of a range of bile acids selected for their varying physicochemical properties. The endpoints were catalytic activity of three individual CYP proteins, CYP 2A1 (measured as testosterone 7α-hydroxylase activity), 2C11 (testosterone 2α-hydroxylase and 16α-hydroxylase) and 3A2 (testosterone 6β-hydroxylase), and the non-CYP enzymes, steroid 17β-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum following bile duct ligation, there was a significant reduction in the activity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-dependent and there was no difference in inhibitory activity towards the male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A1 and other microsomal enzymes. Taurocholic acid was twice as potent an inhibitor as unconjugated cholic acid, the respective apparent I₅₀ values being ～0.6 mmol/L compared with ～1.2 mmol/L. The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid, were also more potent inhibitors than cholic acid, exhibiting I₅₀ values in the range of 0.3–0.5 mmol/L, but the monohydroxy bile acid, lithocholic acid, was the most potent inhibitor (I₅₀～0.2 mmol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activity of microsomal enzymes in livers of bile duct-ligated rats and indicate that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the male sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive mechanism.     

Chronic dietary iron overload in rats results in impaired calcium sequestration by hepatic mitochondria and microsomes [corrected

The purpose of these experiments was to determine whether chronic dietary iron overload causes impairment of hepatic mitochondrial and/or microsomal calcium sequestration. Experimental iron overload was produced by feeding three groups of rats a chow diet supplemented with 3.0% (wt/wt) carbonyl iron for up to 8 weeks achieving graded increases in hepatic iron concentrations ranging from 1360 to 3170 micrograms/g. At low levels of iron overload, there were no changes in mitochondrial oxidative metabolism or calcium sequestration, whereas at moderate and high degrees of iron loading, both of these parameters were significantly reduced. In contrast, there were significant decreases in microsomal cytochrome P450 levels and microsomal calcium sequestration at all three levels of iron loading. These abnormalities occurred at hepatic iron concentrations at which the authors have previously found evidence of hepatic organelle lipid peroxidation. These alterations in organelle calcium sequestration may impair intracellular calcium homeostasis in the liver and contribute to subsequent cellular injury.     

Magnetic Circular Dichroism Studies XXXV. A Comparison of Cytochromes P-450 and P-448

Cytochromes P-450 and P-448 in microsomal suspensions have been shown to be spectrally distinct by magnetic circular dichroism spectroscopy. Furthermore, this technique can be used to measure induction of these two cytochromes by phenobarbital and 3-methyl-cholanthrene. Magnetic circular dichroism spectroscopy is thus at least as useful as difference spectroscopy for the investigation of P-450 and P-448 and more informative because the presence of cytochrome b₅ and hemoglobin can be detected concurrently. We have also shown that the molar magnetic ellipticity for reduced + CO treated cytochrome P-450 of Pseudomonas grown on camphor is of similar value to that of reduced + CO treated microsomal P-450 and P-448.