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.     

Age-related changes in renal metabolism of acetaminophen in male Fischer 344 rats

Male Fischer 344 rats classified as young (2–4 months), middle age (12–15 months) and aged (22–25 months) were examined for changes in renal metabolism of acetaminophen. Renal microsomal cytochrome P-450 levels were 30% and 48% lower in middle age and aged animals, respectively, compared to young; however, no age-related changes were observed in NADPH-cytochrome c reductase activity. Renal mixed-function oxidase production of the reactive intermediate of acetaminophen was reduced 50% in aged rats. The deacetylation of acetaminophen to the nephrotoxic metabolite p-aminophenol by the renal 10,000 × g supernatant fraction was not changed by age. Thus, p-aminophenol becomes the proportionately greater nephrotoxic metabolite of acetaminophen produced by the aged rat kidney. Whole kidney reduced and oxidized glutathione content tended to be higher in aged rats, while glutathione S-transferase activity was 46% lower in aged animals. These results demonstrate that age alters several indices of renal metabolism of acetaminophen in male Fischer 344 rats.     

Qualitative changes in rat liver microsomal NADPH cytochrome C (P-450) reductase during aging

The hepatic drug-metabolizing capacities of rodents and humans undergo marked age-dependent declines, particularly between maturity and senescence. This decline probably results from distinct qualitative and quantitative changes in the microsomal mixed function oxidase system. Previous studies reported age-related reductions in the amount of smooth surfaced endoplasmic reticulum membrane and the specific activities of several constituent enzymes of this drug-metabolizing system. The present study has subjected one of these enzymes, NADPH cytochrome c (P-450) reductase, to an extensive analysis and some of these data are reported here. The microsomal membranes of young Fischer 344 male rats (3 months) yielded approximately twice the enzyme recovered from those of old animals (27 months). Furthermore, the specific activity of the purified enzyme isolated from young animals was two-fold higher than that obtained from senescent rats. The “old” enzyme also exhibited an increased thermostability profile in comparison to the “young” enzyme. These data provide the first evidence of possible molecular alterations responsible for the well-documented age-dependent decline in the functional capacity of the rat liver microsomal mixed function oxidase system.     

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.     

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.     

Differences in Hepatic Microsomal Cytochrome P-450 Isoenzyme Induction by Pyrazole, Chronic Ethanol, 3-Methylcholanthrene, and Phenobarbital in High Alcohol Sensitivity (HAS) and Low Alcohol Sensitivity (LAS) Rats

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heat and detergent on glucose-6-phosphatase activity in microsomes isolated from rats of different ages

Hepatic microsomal glucose-6-phosphatase activity in male Fischer rats declines with age. It is unknown whether the phospholipid composition of the microsomal membrane contributes to this age-related decline in enzyme activity. This report compares the effect of two lipid modifiers, heat and detergent, on the activity of glucose-6-phosphatase in microsomes isolated from male Fischer 344 rats of different ages. The heat lability of this enzyme in intact and detergent-disrupted microsomes was not age dependent. These results suggest that the decline in glucose-6-phosphatase activity during liver senescence is not due to lipid changes in the microenvironment of this enzyme that are detectable by thermolability and detergent sensitivity.     

Hepatic mitochondrial cytochrome P-450: Isolation and functional characterization

A CO-binding heme protein was solubilized and partially purified from the inner membrane fraction of rat liver mitochondria by a modification of a method [Imai, Y. & Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14] developed to purify cytochrome P-450 from liver microsomes. The partially purified preparation contained protoheme and its spectral properties are characteristic of the heme proteins of the cytochrome P-450 family. The isolated cytochrome P-450 preparation could reconstitute a CO-sensitive, NADPH-dependent 26-hydroxylation activity for 5beta-cholestane-3alpha,7alpha,-12alpha-triol when supplemented with NADPH-adrenodoxin reductase and adrenodoxin, both purified from bovine adrenocortical mitochondria. Unlike a cytochrome P-450 purified from liver microsomes of drug-untreated rats, however, the liver mitochondrial cytochrome P-450 could not catalyze NADPH-dependent benzphetamine N-demethylation in the presence of adrenodoxin reductase and adrenodoxin or function with the purified microsomal NADPH-cytochrome c reductase plus Emulgen 913 as an electron-donating system. It is concluded that the rat liver inner mitochondrial membrane houses a species of cytochrome P-450 functional in 5beta-cholestane-3alpha,7alpha,12alpha-triol 26-hydroxylation.     

Age-dependent changes in rat liver microsomal NADPH cytochrome C (P-450) reductase: a kinetic analysis

Rat liver microsomal NADPH cytochrome c (P-450) reductase (EC 1.6.2.4) exhibits several marked age-dependent changes, including a decline in specific activity, reduced inducibility and enhanced thermostability, which are indicative of an alteration in the quality of this enzyme. The present study examined the kinetic profile of the microsomal-bound enzyme in an effort to further define the effects of aging on the hepatic mixed function oxidase system. Intact microsomes isolated from young adult (3 months), mature (16 months) and senescent (27 months) rats were subjected to an extensive double reciprocal kinetic analysis employing NADPH and cytochrome c as substrates. The Km values obtained with the initial substrate (NADPH) remained unchanged with animal age, whereas there was a decline in this parameter for the artificial acceptor substrate, cytochrome c. The Vmax values for both substrates were reduced as a function of increasing age, perhaps reflecting a concomitant decline in the relative amount(s) of efficient reductase in the microsomes.     

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.     

Effects of frying oil and Houttuynia cordata thunb on xenobiotic-metabolizing enzyme system of rodents

AIM: To evaluate the effects of frying oil and Houttuynia cordata Thunb (H. cordata), a vegetable traditionally consumed in Taiwan, on the xenobiotic-metabolizing enzyme system of rodents. METHODS: Forty-eight Sprague-Dawley rats were fed with a diet containing 0%, 2% or 5% H. cordata powder and 15% fresh soybean oil or 24-h oxidized frying oil (OFO) for 28 d respectively. The level of microsomal protein, total cytochrome 450 content (CYP450) and enzyme activities including NADPH reductase, ethoxyresorufin 0-deethylase (EROD), pentoxyresorufin 0-dealkylase (PROD), aniline hydroxylase (ANH), aminopyrine demethylase (AMD), and quinone reductase (QR) were determined. QR represented phase Ⅱ enzymes, the rest of the enzymes tested represented phase Ⅰ enzymes. RESULTS: The oxidized frying oil feeding produced a significant increase in phase Ⅰ and Ⅱ enzyme systems, including the content of CYP450 and microsomal protein, and the activities of NADPH reductase, EROD, PROD, ANH, AMD and QR in rats (P<0.05). In addition, the activities of EROD, ANH and AMD decreased and QR increased after feeding with H. cordata in OFO-fed group (P<0.05). The feeding with 2% H. cordata diet showed the most significant effect. CONCLUSION: The OFO diet induces phases I and II enzyme activity, and the 2% H. cordata diet resulted in a better regulation of the xenobiotic-metabolizing enzyme system.     

Altered drug metabolism in hepatic and extrahepatic tissues in mice as a function of age

An increase in the susceptibility to drugs is known to occur with advanced age. Several possible explanations for this phenomenon exist, but the exact cause has not been determined. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (ECD) and aniline hydroxylase activities of hepatic and extrahepatic tissues as well as hepatic cytochrome P-450 content of female Swiss Webster mice at 1, 3, 6, 9, 12, 15 and 18 months of age were examined. AHH activity in liver and intestine reached a maximum at 6 mo. of age and began to decline thereafter. No change in pulmonary AHH activity was observed with age. ECD activity in liver as well as lungs was maximal at 6 mo. of age and decreased thereafter. Hepatic aniline hydroxylase activity exhibited over a 700% increase between one to six mo. of age, and declined by approximately 52% by 15 mo. of age as compared to peak activity. Hepatic cytochrome P-450 content was highest at 3 mo. of age and significantly decreased by 12 mo. The results demonstrate that decreases in microsomal mixed function monooxygenase activities do occur with advanced age and may contribute to a decreased rate of metabolism and increased susceptibility to drugs and other foreign chemicals with age.     

Lifelong caloric restriction and interleukin-6 secretion from adipose tissue: effects on physical performance decline in aged rats

We investigated whether caloric restriction (CR) improves physical performance in a rodent model of aging, and whether this effect is accompanied with a decrease in visceral adipose tissue production of proinflammatory cytokines. Body composition, standardized physical performance measures, as well as in vitro visceral adipose tissue cytokine secretion and circulating levels of an inflammatory marker were cross-sectionally assessed in ad libitum (AL)-fed and lifelong CR Fischer 344 x Brown Norway male rats aged 18, 24, and 29 months. Fat to lean mass ratio increased and physical performance declined with age in the AL rats. Compared to AL rats, CR rats had lower fat mass, fat to lean ratio, adipose tissue secretion of interleukin-6, and circulating levels of C-reactive protein, and higher physical performance scores. Therefore, CR may be an effective intervention for improving functional status into advanced age and is perhaps mediated via a reduction in adipose tissue-generated proinflammatory cytokine production.     

Long-term maintenance of the adult pattern of liver-specific expression for P-450b, P-450e, albumin and alpha-fetoprotein genes in intrasplenically transplanted hepatocytes.

Hepatocytes isolated from livers of Fischer 344 rats and transplanted into the spleens of rats from the same strain survived for at least 15 mo in the absence of immunosuppressive drugs. Hepatocytes attached themselves only in the red pulp of the spleen, most commonly in clumps without a discernible structure. Throughout the 15-mo period, intrasplenically transplanted hepatocytes expressed cytochrome P-450b, P-450e and albumin messenger RNAs, whereas alpha-fetoprotein messenger RNA was not expressed. In addition, the relative expression of albumin and P-450 genes was similar to that in liver. For example, albumin messenger RNA was expressed to higher levels than P-450b or e messenger RNAs. Northern blots hybridized with oligonucleotides specific for P-450b or P-450e showed that, as in liver, both P-450b and P-450e genes were induced in response to phenobarbital. Quantitative slot-blot hybridizations performed at 15 days and 1, 6, and 15 mo after hepatocyte transplantation revealed that cytochrome P-450b and P-450e messenger RNAs were induced about 20- to 30-fold by a single dose of phenobarbital. This level of induction was also similar to that observed in liver. Hence, intrasplenically transplanted hepatocytes represent a unique system in which hepatocytes, cultured in an extrahepatic in vivo environment, maintain for at least 15 mo a pattern of expression for these four liver genes similar to that in the adult liver. Moreover, these studies suggest that neither the organization of liver into acini nor a specific zonal sinusoidal microenvironment is necessary for adult hepatocytes to respond to phenobarbital with induction of P-450b and P-450e genes.     

Immunofluorescent determination of the lobular distribution of a constitutive form of hepatic microsomal cytochrome P-450

We have used an immunohistochemical approach to study the lobular distribution of constitutive liver microsomal cytochrome P-450. Cytochrome P-450 isolated (10.3 nmoles per mg protein) from hepatic microsomes from untreated, mature male Sprague-Dawley rats was used to produce antisera in rabbits. The IgG fraction produced single precipitin lines of identity with liver microsomes after double immunodiffusion, precipitated 80% of the total microsomal cytochrome P-450 and inhibited three cytochrome P-450-dependent enzyme activities. By these criteria, the IgG appeared to be specific for a constitutive form (or immunochemically related family) of liver microsomal cytochrome P-450. The pattern of fluorescence after indirect immunofluorescent labeling of liver sections depended on the route of tissue preparation and the concentration of primary antibody. In frozen sections, the labeling was uniform throughout the lobule, whereas in "antigen-depleted" paraffin-embedded sections it was heaviest in the centrilobular and midzonal regions. Increasing the concentration of primary antibody to 500 micrograms per ml inhibited the centrilobular labeling in frozen sections in a concentration-dependent manner. When specific isozymes of cytochrome P-450 were induced with phenobarbital or 3-methylcholanthrene, the constitutive cytochrome P-450 was localized predominantly in the periportal region. Decreases in cytochrome P-450 in rats treated with carbon tetrachloride or 1,2-dibromo-3-chloropropane were associated with antigen loss only in necrotic cells. Regional differences in the loss of antigen in paraffin sections and the inhibition of fluorescence in frozen sections establish that the lobular distribution of constitutive hepatic microsomal cytochrome P-450 is qualitatively heterogeneous and may be altered during hepatocellular responses to chemical treatment.     

Cobalt Induction of Hepatic Heme Oxygenase; with Evidence That Cytochrome P-450 Is Not Essential for This Enzyme Activity

Treatment of rats in vivo with cobalt chloride stimulated heme oxidation by hepatic microsomes to levels up to 800% above controls. This treatment also caused increases in liver weight and in total microsomal protein; in contrast, marked decreases were produced in microsomal oxidation of ethylmorphine (80%), and in cytochrome P-450 (60-70%) and heme (30-50%) contents. Cobalt chloride treatment did not affect heme oxidation by the spleen heme oxygenase system.The rate of heme oxidation by hepatic microsomal enzymes and the microsomal content of cytochrome P-450 were found to be unrelated. This conclusion was reached from studies in which microsomal heme oxygenase activity from cobalt-treated animals could be increased by 900% above control levels in the same microsomal preparation in which cytochrome P-450 content was decreased to spectrally unmeasurable amounts after incubation with 4 M urea. The same treatment eliminated ehtylmorphine demethylation and decreased microsomal NADPH-cytochrome c reductase (EC 1.6.2.4) activity by 75%.It is concluded that (i) the hepatic microsomal enzyme system that oxidizes heme compounds is not the same as that which metabolizes drugs, (ii) cytochrome P-450 is not essential for the oxidation of heme by liver cells, (iii) there is no direct relationship between the rate of heme oxidation and the level of NADPH-cytochrome c reductase activity, and (iv) the oxidation of heme is protein-dependent and the active proteins are inducible, but are different from those involved in drug metabolism.     

Effect of alpha-tocopherol on hepatic mixed function oxidases in hepatic ischemia/reperfusion.

This study was done to determine the relationship between microsomal lipid peroxidation during hepatic ischemia/reperfusion and alteration in cytochrome P-450-dependent drug metabolism. Rats were pretreated with alpha-tocopherol to inhibit lipid peroxidation or with vehicle (soybean oil) and then subjected to 60 min no-flow hepatic ischemia in vivo. Control animals were time-matched sham-ischemic animals. After 1, 5 or 24 hr of reperfusion, liver microsomes were isolated and cytochrome P-450 and mixed function oxidases were studied. In vehicle-treated ischemic rats, serum ALT levels peaked at 5 hr (5,242 +/- 682 U/L) and were significantly reduced by alpha-tocopherol pretreatment (1,854 +/- 229 U/L, p less than 0.01). Similarly, microsomal lipid peroxidation was elevated in the vehicle-treated ischemic group, but this elevation was prevented by alpha-tocopherol pretreatment. Microsomal cytochrome P-450 content and aminopyrine-N-demethylase activity were both decreased in vehicle-treated ischemic rats to 60% and 70% of sham-ischemic control levels, respectively. Although alpha-tocopherol restored cytochrome P-450 content to the level of sham-ischemic control rats, aminopyrine-N-demethylase activity remained at 76% of control with alpha-tocopherol treatment (p less than 0.01 compared with sham-ischemic control). In contrast to what was seen with cytochrome P-450 and aminopyrine-N-demethylase, aniline p-hydroxylase activity was elevated in the vehicle-treated ischemic rats compared with sham-ischemic control rats. These increases were prevented by alpha-tocopherol pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of aging on the metabolism of p-nitroanisole and p-nitrophenol in isolated rat hepatocytes

The effects of aging on the monooxygenation of p-nitroanisole (PNA) and conjugation of p-nitrophenol (PNP) were determined in hepatocytes isolated from young (2–4 months) and old (20–24 months) male Fischer 344 rats. The rate of PNA O-demethylation was significantly decreased (41%) in aged rats. PNP formed from the monooxygenation of PNA was predominantly conjugated by suIfation. The decrease in monooxygenation of PNA was accompanied by a 57% decrease in the rate of PNP-sulfate production. No change in the glucuronidation of PNP or level of unconjugated PNP was observed during aging when PNA was used as substrate. With PNP as substrate, glucuronidation was the major conjugative pathway. An age-related decrease (70%) in the suIfation capacity was observed at both 100 and 200 μM PNP, but no change in the glucuronidation of PNP was seen in aged rats. Therefore, the overall conjugation of PNP was decreased by 30% with age. Microsomal glucuronyltransferase activity toward PNP was unaffected by aging, and microsomal cytochrome P-450 levels were reduced 34% in aged rats. This study demonstrates age-related decreases in PNA monooxygenation and PNP suIfation in isolated rat hepatocytes, while PNP glucuronidation capacity is unaffected during aging.     

Influence of aging on ethanol and acetaldehyde oxidation in female rat liver

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.     

Enhanced drug-metabolizing capacity within liver adjacent to human and rat liver tumors.

Cytochrome P-450 content (nmol/g of liver) differed within regions of rat liver according to proximity to intrahepatically implanted Morris hepatoma 7795 or 5123D. Liver adjacent to tumor had higher microsomal cytochrome P-450 content, decreased DNA content (mg/g of liver), and unaltered cytochrome c reductase activity compared to histologically indistinguishable liver far-removed from the tumor. Liver either adjacent to or far-removed from tumor contained markedly more cytochrome P-450 and higher cytochrome c reductase activity but less DNA than transplanted Morris hepatomas 7795 and 5123D that were grown intrahepatically. Compared to intramuscular implants of these same tumors, intrahepatically implanted Morris hepatomas 7795 and 5123D had increased cytochrome P-450 content. Tumor-containing liver from two human subjects revealed regional changes in cytochrome P-450-mediated monooxygenases similar to those observed in rats. These results suggest that histomorphically nontumorous mammalian liver directly adjacent to intrahepatic tumors exhibits previously unsuspected biochemical alterations.