Microsomal Cytochrome P-450 Levels Research Articles

Antidotal Effect of Lipopolysaccharide Against Acetaminophen‐Induced Mortality in Mice

The possibility that pretreatment with LPS (lipopolysaccharide obtained from Escherichia coli), an immune system stimulant and interferon inducer, could prevent the hepatotoxic effects of acetaminophen (NAPAP) was investigated in mice. When mice were pretreated with LPS (4 mg/kg), intraperitoneally for 24 hr the mortality caused by NAPAP was considerably reduced. Histological examination of the livers and leakage of the enzymes into the blood demonstrated that NAPAP-induced necrosis was decreased in LPS-treated mice compared to that induced by NAPAP alone. Pretreatment with 400 mg/kg or 800 mg/kg of NAPAP decreased the amount of covalently-bound acetaminophen metabolites. Since the level of hepatic glutathione and microsomal cytochrome P-450 were depressed in these experiments, it is concluded that LPS depresses the cytochrome P-450 species responsible for the formation of the toxic metabolites and that less reactive species are available for binding to cell macromolecules.

Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats.

Cytochrome P450 content and activities are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared with those of normotensive, Wistar-Kyoto (WKY), control rats during the period of rapid elevation of blood pressure. We studied the effect of heme arginate, a potent inducer of heme oxygenase (EC 1.14.99.3), on microsomal cytochrome P450 levels and activities and blood pressure in SHR at 7 wk of age. Administration of heme arginate (15 mg/kg body weight for 4 d) resulted in a marked decrease in blood pressure from 156.3 +/- 4.7 to 129.8 +/- 4.5 mm Hg (P less than 0.001), whereas blood pressure in SHR receiving the vehicle control was not affected. The blood pressure of age-matched WKY was not affected by heme arginate. Heme oxygenase activity increased in both hepatic and renal microsomes of SHR and WKY by two- to four-fold after treatment with heme arginate. Maximal increase of heme oxygenase mRNA occurred 5-7 h after the last injection of heme arginate and returned to control levels after 24 h. The increase in heme oxygenase activity was associated with a parallel decrease in cytochrome P450 content and in the activity of cytochrome P450 omega/omega-1 arachidonate hydroxylases in kidneys of SHR. It is postulated that heme arginate treatment resulted in induction of heme oxygenase which consequently led to a diminution of cytochrome P450, especially the arachidonate omega/omega-1 hydroxylases leading to a marked decrease in 19-hydroxyeicosatetraenoic acid (HETE) and 20-HETE. The effect of heme arginate on blood pressure may be mediated via these biochemical events inasmuch as both 19-HETE and 20-HETE produced by the kidney may promote hypertension by causing vasoconstriction and sodium retention.

Interleuskins-1 and -6 differently affect serum α1-acid glycoprotein and hepatic microsomal cytochrome P-450 levels in the rat

Interleuskins-1 and -6 differently affect serum α1-acid glycoprotein and hepatic microsomal cytochrome P-450 levels in the rat

Hydroxylation of primisulfuron by an inducible cytochrome P450-dependent monooxygenase system from maize

Microsomes were prepared from etiolated maize seedlings and incubated with [ 14C]primisulfuron (2-[3-(4,6-bis(difluoromethoxy)-pyrimidin-2-yl)-ureidosulfonyl]-benzoic acid methylester). Two enzymatic reaction products were formed in the presence of O 2 and NADPH. Comparison on high-performance liquid chromatography with synthetic reference standards and mass spectrometry of the two in vitro metabolites revealed that [ 14C]primisulfuron was hydroxylated at two different sites, i.e., at the phenyl and at the pyrimidine ring, respectively. Both hydroxylation reactions were inhibited in vitro by tetcyclacis as well as by CO in the presence of O 2. CO inhibition was reversed by irradiation of the reaction mixture with white light. Enzyme activities were localized predominantly in the shoots. Apparent K m values for [ 14C]primisulfuron were estimated to be 137 and 47 μ M, and V max to be 427 and 261 pmol/hr/mg protein, for the hydroxylation on the pyrimidine and phenyl ring, respectively. Formation of these metabolites from [ 14C]primisulfuron was barely detectable in microsomes from germinating seedlings. However, seed treatment (0.2% w w ) with the safener, CGA 154281 (4-[dichloroacetyl]-3,4-dihydro-3-methyl-2 H-1,4-benzoxazine), increased microsomal cytochrome P450 levels twofold and dramatically stimulated in vitro [ 14C]primisulfuron phenyl- and pyrimidine-ring hydroxylation. From these data it is concluded that hydroxylation of primisulfuron in maize microsomes is catalyzed by an inducible cytochrome P450 monooxygenase system.

Effect of dietary fat on the induction of hepatic microsomal cytochrome P450 isozymes by phenobarbital

Dietary polyunsaturated fatty acid is needed for optimal induction of cytochrome P450. In this study we quantitated cytochrome P450 hemoproteins in male Sprague-Dawley rats that were starved for 36 hr and then refed a fat-free diet (FF) or a diet containing 20% corn oil for 4 days. Some received phenobarbital (Pb) sodium (80 mg/kg, i.p., daily) for 3 days prior to decapitation. Microsomal cytochrome P450 levels were measured by carbon monoxide binding spectra, and the P450 isozymes separated by sodium dodecyi sulfate-polyacrylamide gel electrophoresis were quantitated by gel scanner. Cytochrome P450 PB-B was quantitated by a Western blot technique. Rats fed FF diet and administered Pb had only 21% more microsomal P450 than non-induced controls, whereas rats fed 20% corn oil diet had 59% more P450 and Pb-treated rats fed 20% corn oil diet had 181% more P450 than FF controls. Analysis of gels showed 32, 59 and 124% more P450 protein, respectively, in FF Pb, corn oil control or corn oil Pb groups than in FF controls. Cytochrome P450 PB-B was not detected in non-induced groups. but quantitation by Western blot yielded 0.32 and 0.70 nmol/mg protein, respectively, in FF Pb and corn oil Pb groups. Our findings suggest that deprivation of dietary fat reduces the total amount of cytochrome P450 hemoprotein and its inducibility by Pb through decreased P450 hemoprotein synthesis. The limiting factor(s) restricting synthesis of new cytochrome P450 hemoproteins in rats refed a diet devoid of fat may be the inability to respond to the inducer (Pb) or the paucity of utilizable fatty acids needed for synthesis of the phospholipid matrix of the endoplasmic reticulum necessary for the support and proper juxtapositioning of these protein molecules.

Hepatic microsomal protein and cytochrome P‐450 in BALB/c mice infected with Leishmania

The effects of infection of mice with Leishmania major on liver microsomal protein and cytochrome P-450 were examined. The levels of hepatic microsomal protein and cytochrome P-450 were monitored at 6, 7, 9 and 12 weeks post-infection. The results indicated that the amount of hepatic microsomal protein and cytochrome P-450 were unchanged throughout the course of infection with L. major, despite the high degree of parasite proliferation in Kupffer cells and marked reduction in phagocytosis. The current results clearly indicate that Leishmania-induced macrophage suppression has no inhibitory effect on hepatic microsomal protein and cytochrome P-450.

Effects of Chronic Ethanol Consumption on Carcinogen Activating and Detoxifying Systems in Rat Upper Alimentary Tract Tissue

The effect of chronic ethanol consumption on enzyme systems directly involved in carcinogen activation and detoxification were studied in rat upper alimentary tract tissue. Microsomal cytochrome P-450 (P-450) levels and glutathione levels as well as glutathione transferase and UDP-glucuronic acid transferase (UDPGT) activities were measured in mucosa scraped from esophagus, forestomach and glandular stomach of rats which had been pair-fed ethanol or dextrimaltose-containing diets. Esophageal and forestomach P-450 levels were increased in the ethanol-fed rats. The ethanol diet also produced a small but significant increase in esophageal glutathione transferase levels. Glutathione levels and UDPGT activity were unaffected. Since P-450 is directly involved in the activation of many chemical carcinogens, these results are consistent with the hypothesis that the increase in upper alimentary tract cancer risk associated with alcohol abuse is due, at least in part, to ethanol's altering the balance between carcinogen activation and detoxification.

Depression of cytochrome P450-dependent drug biotransformation by poly(rI·rC) and aspirin

The inhibition of hepatic microsomal cytochrome P450 and cytochrome b5 levels by poly(rI.rC) and aspirin in vitro was studied in male Swiss mice. Poly(rI.rC) (10 mg/kg) decreases cytochrome P450 to a level of 61% and cytochrome b5 to 31%, while the activity of aminopyrine demethylase was decreased to 66%. Aspirin (200 mg/kg) decreased the level of cytochrome P450 and cytochrome b5 to 45 and 23%, respectively. Further studies on the effect of poly(rI.rC) showed that this decrease in the levels of cytochromes was mainly due to alteration in the protein synthesis of these hemoproteins.

Hepatotoxicity of pulegone in rats: Its effects on microsomal enzymes, in vivo

Oral administration of pulegone (400 mg/kg) to rats once daily for five days caused significant decreases in the levels of liver microsomal cytochrome P-450 and heme. Cytochrome b 5 and NAD(P)H-cytochrome c-reductase activities were not affected. Massive hepatotoxicy accompanied by an increase in serum glutamate pyruvate transaminase (SGPT) and a decrease in glucose-6-phosphatase were observed upon treatment with pulegone. A significant decrease in aminopyrine N-demethylase was also noticed after pulegone administration. Menthone or carvone (600 mg/kg), compounds related to pulegone, when administered orally did not cause any decrease in cytochrome P-450 levels. The hepatotoxic effects of pulegone were both dose and time dependent. Pretreatment of rats with phenobarbital (PB) or diethylmaleate (DEM) potentiated the hepatotoxicity caused by pulegone, whereas, pretreatment with 3-methylcholanthrene (3-MC) or piperonyl butoxide protected from it. It appears that a PB induced cytochrome P-450 catalysed reactive metabolite(s) may be responsible for the hepatotoxicity caused by pulegone.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Developmental Changes in the Microsomal Monooxygenase System and the In Vivo Metabolism of Aldrin in Larvae of the Mexican Bean Beetle (Coleoptera: Coccinellidae)

In vivo metabolism of topically applied aldrin and the specific levels of whole body and abdominal microsomal cytochrome P-450, P-420, b5 and creductase were measured during development in the penultimate and last instar of the Mexican bean beetle, Epilachna varivestis Mulsant. There were no significant differences between molting larvae (from the penultimate to the last instar), last-instar feeding larvae and prepupae in these monooxygenase properties for whole-body microsomal preparations, and in respect to in vivo metabolism. Cytochrome P-450, b5, and creductase were higher in the abdominal microsomes of feeding-stage larvae than in whole-body microsomes from the same stage, and cytochrome P-450 and b5 were higher in the abdominal microsomes of feeding-stage larvae than in the abdominal microsomes from prepupae. The molar ratio of P-450 to P-420 was constant (approximately 1) for all microsomal preparations examined. The high tolerance of Mexican bean beetle larvae to topically applied aldrin could not be predicted based on poor penetration or the inability of aldrin to be epoxidized to dieldrin.

Induction of the rat hepatic microsomal mixed-function oxidases by 3 imidazole-containing antifungal agents: selectivity for the cytochrome P-450IIB and P-450III families of cytochromes P-450

Administration of the imidazole antifungal agents ketoconazole, miconazole and clotrimazole gave rise to increases in the microsomal cytochrome P-450 levels and the NADPH-dependent reduction of cytochrome c. Clotrimazole, and to a much lesser extent miconazole and ketoconazole, stimulated the dealkylation of pentoxyresorufin. All 3 agents gave rise to small, but significant increases in the O-deethylations of ethoxycoumarin and ethoxyresorufin. The antifungal-induced O-deethylation of ethoxycoumarin was much more sensitive to inhibition by metyrapone rather than by α-naphthoflavone. The binding of metyrapone to reduced microsomes was enhanced by treatment of animals with the 3 antifungal agents, clotrimazole being clearly the most potent. Immunoquantitation of cytochrome P-450 proteins using an ELISA procedure and employing anti-cytochrome P-450c ( P-450IA1, P-448 low spin) and P-450b ( P-450IIB1) antisera revealed that clotrimazole and miconazole, but not ketoconazole, induced the levels of phenobarbital-induced cytochromes P-450, while none of the antifungal agents increased the levels of cytochrome of P-448 proteins. Similar results were obtained using Western blots employing the above antibodies. On SDS-polyacrylamide gel electrophoresis microsomes derived from animals pretreated with clotrimazole showed intensification of a band at 51 kDa which was identified by Western blotting as the PCN-inducible form of cytochrome P-450 (cytochrome P-450p, P-450III family). Similar, but less pronounced intensification was seen with microsomes from animals pretreated with miconazole and ketoconazole. Furthermore, microsomes from clotrimazole- and ketoconazole-treated animals interacted with erythromycin to yield type I spectra. It is concluded that the imidazole-containing agents clotrimazole and miconazole, and to a much lesser extent ketoconazole, are potent inducers of the rat hepatic microsomal mixed-function oxidases, displaying selectivity towards the P-450IIB (phenobarbital-inducible) and P-450III (PCN-inducible) families of cytochrome P-450 proteins.

Effect of chronic aurothioglucose treatment of rats on kidney catalase and cytochromes

Catalase activity and cytochrome content were measured in kidneys of Fisher 344 rats injected with aurothioglucose (ATG) either daily for 3 days or 5 days a week for up to 8 wk. Catalase activity was decreased 39%, 59%, and 48% (all p < 0.001) after 3 days, 2 wk, and 8 wk, respectively. Microsomal cytochrome P-450 levels decreased 71%, 86%, and 80% (all p < 0.001) after 3 days, 2 wk, and 8 wk, respectively. In contrast, cytochrome b 5 was significantly increased at 3 days and 2 wk, but not at 8 wk. Microsomal heme contents decreased 44% ( p < 0.001), 34% ( p < 0.001), and 22% ( p > 0.05) at 3 days, 2 wk, and 8 wk, respectively. The content of mitochondrial cytochromes aa 3, b, c 1, and c were not affected after 8 wk of ATG treatment. In vitro inhibition of the heme-containing enzyme δ-aminolevulinic acid dehydratase by ATG was reversible in the presence of physiological concentrations of small thiols. Although the activity of this enzyme in kidneys of ATG-treated rats was not measured, its significant inhibition in vivo by ATG appears unlikely. This study demonstrates that there were differential effects of gold on the various cytochromes and that changes in catalase activity paralleled changes in cytochrome P-450 and heme contents in the kidneys of ATG-treated rats. The findings are relevant to nephrotoxicity during chrysotherapy.

An immunotoxicological evaluation of 4,4′-thiobis-(6- t-butyl- m-cresol) in female B6C3F1 mice : 1. Body and organ weights, hematology, serum chemistries, bone marrow cellularity, and hepatic microsomal parameters

Adult female B6C3F1 mice were gavaged with 4,4′-thiobis-(6- t-butyl- m-cresol) (TBBC) in corn oil at doses of 10, 100, or 200 mg/kg daily for 14 consecutive days. There was no overt toxicity, as manifested by grossly observable behavioral changes, decreased growth rate over the exposure period, or mortality. There were also no marked effects on serum chemistries or hematology, with the exception of a significant increase (41%) in the number of leukocytes at the highest dose. Absolute differential counts indicated that significant increases occurred in the number of lymphocytes (31%) and neutrophils (177%). Studies with bone marrow indicated a significant 30% increase in the number of cells/femur from animals treated with the highest dose of TBBC. The number of macrophage progenitors (CFU-M)/femur was significantly increased by 28%, while the number of granulocyte-monocyte progenitors (CFU-GM)/femur was nonsignificantly increased by 20% in the high dose animals. The weight of both the spleen and liver was increased in a dose-related fashion, although the histopathology of the spleen of TBBC-treated mice was not different from control. The livers of mice receiving the high dose showed mild focal hydropic degeneration, mild hepatitis, and a slight increase in the number of Kupffer cells. No other organs were affected. Liver microsomal protein and cytochrome P-450 levels were increased in a dose-related fashion. Enzyme activities of aminopyrine demethylase and aniline hydroxylase, but not arylhydrocarbon hydroxylase, were also increased in a dose-related fashion.

Biosynthesis of pyrimidine nucleotides and level of cytochrome P-450 in rat liver and kidney after clofibrate administration (an in vivo study).

Like other lipid-soluble xenobiotics, clofibrate (ethyl-2-(4-chlorophenoxy)-2-methylpropanoate) increased the level of microsomal cytochrome P-450 in liver and decreased the utilization of 14C-orotic acid for the synthesis of hepatic cytidine nucleotides. This phenomenon was associated with the increased (a) uptake of 14C-cytidine, (b) total content of cytidine components of the acid-soluble extract and (c) utilization of this nucleoside for the synthesis of RNA. No changes were observed in uridine components. Clofibrate also increased the level of cytochrome P-450 in kidney microsomes; the degree of induction was almost the same as in the liver. The variations of renal pyrimidine metabolism after administration of the drug were analogous to those observed in the liver.

Selective inhibition of 14 alpha-desmethyl sterol synthesis in Candida albicans by terconazole, a new triazole antimycotic.

Terconazole, a new broad spectrum antimycotic triazole derivative, has been shown to have potent activity against Candida albicans in vitro and to be effective in animal models of yeast infections. The present study explored a possible mechanism of anticandidal activity of terconazole. The compound inhibited production of 14 alpha-desmethyl sterols (e.g. ergosterol) in C. albicans at concentrations (IC50 = 3-6 x 10(-9) M) lower than those inhibiting the in-vitro growth of the yeast. There was concomitant accumulation of methylated sterols, (e.g. lanosterol), which are considered detrimental to normal yeast cell membrane function. Terconazole stimulated incorporation of 14C-acetate into triglycerides, but had no other effect on C. albicans lipid metabolism. At concentrations greater than or equal to 10(-6)M terconazole inhibited the oxidation of 14C-acetate into 14CO2 in C. albicans although the mechanism for this effect remains unclear. These data indicate that terconazole is a specific inhibitor of yeast C-14 desmethyl sterol production in C. albicans. Furthermore, terconazole reduced cytochrome P-450 levels in yeast microsomes at concentrations 10,000-fold below those at which it showed effects on rabbit liver microsomes. These data indicate a species specificity for the biochemical actions of terconazole. The C-14 alpha-desmethylase system in yeast cell membranes is cytochrome P-450 associated. Thus, terconazole, was a potent inhibitor of C-14 desmethyl sterol synthesis. This effect could contribute to the anticandidal activity of the drug.

Depression of rat testicular 17-hydroxylase and 17,20-lyase after administration of 2,3,7,8-Tetrachlorodibenzo- p-Dioxin (TCDD)

Toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), such as chloracne, hirsutism, and skin hyperpigmentation, suggest endocrine involvement, however, little is known about the effects of TCDD on steroidogenic organs. It is known that TCDD can cause decreases in testicular heme, testicular microsomal cytochrome P-450, and serum testosterone in the rat. This study was designed to examine the activities of the testicular hemoprotein microsomal cytochrome P-450-dependent enzymes, 17-hydroxylase and 17,20-lyase, following a single, oral dose of either 12.5, 25, or 50 μg/kg TCDD. TCDD caused dose- and time-dependent decreases in the activity of the 17-hydroxylase enzyme. Significant decreases were observed at 3, 7 and 14 days at the lowest dose of 12.5 μg/kg TCDD. The 17,20-lyase enzyme seemed to be less sensitive to the toxic effects of TCDD with significant decreases in enzyme activity being observed at days 3, 7 and 14 only after treatment with 50 μg/kg TCDD. Both microsomal cytochrome P-450 and serum testosterone levels decreased in a dose- and time-dependent manner following 12.5, 25 and 50 μg/kg doses of TCDD. These results indicate that decreased testosterone production following treatment with TCDD is related to decreased activities of the testicular microsomal cytochrome P-450-dependent enzymes 17-hydroxylase and 17,20-lyase.

In vitro effects of trans-4-hydroxy-2-alkenals on mouse liver cytochrome P-450

Under in vitro conditions, trans-4-hydroxy-2-hexenal (t-4HH), trans-4-hydroxy-2-nonenal (t-4-HN) and trans-2-hexenal (t-2H) significantly reduced the levels of mouse liver microsomal cytochrome P-450. Incubation of trans-4-hydroxy-alkenals, under anaerobic conditions in the absence of an NADPH-generating system indicated that these compounds were converting cytochrome P-450 to cytochrome P-420. Prior activation by the mixed function oxidase system was not required for trans-4-hydroxy-alkenals to alter cytochrome P-450 concentrations. trans-4-Hydroxy-alkenals and non-hydroxylated α,β-unsaturated aldehydes may be exerting their effects on cytochrome P-450 by binding to sulfhydryl groups in a similar manner as reported for sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate.

Vitamin A levels in regenerating rat liver. Effects on microsomal drug metabolism.

After being maintained on a vitamin A-deficient or complete diet for a period of five weeks, male Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy (PH) or sham operation. The vitamin A content of the liver of vitamin A-deficient, PH rats was below the limit of detection (less than 1 microgram/g liver). Rats fed the control diet and subjected to PH had hepatic levels of vitamin A that were 37% and 49% lower 48 and 72 hours after surgery, respectively, when compared with sham-operated controls. Hepatic microsomal cytochrome P-450 levels were significantly reduced in PH rats fed the complete diet 48 hours after PH and in PH rats fed either the deficient or complete diet 72 hours after. Vitamin A deficiency alone significantly reduced cytochrome P-450 levels. A combination of vitamin A deficiency and PH had the most dramatic effect on cytochrome P-450 and aminopyrine N-demethylase, which reduced the activity to approximately 50% of the activities found in the sham-operated control group. PH resulted in the greatest reduction in the rate of disappearance of benzo[a]pyrene in the presence of liver microsomes prepared from vitamin A-deficient rats.

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.