Mechanisms of Ageing and Development, 60 (1991) 61-74
61
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF A G I N G AND O T H E R F A C T O R S ON M O N O C Y T E H Y D R O C A R B O N H Y D R O X Y L A S E ACTIVITY
ARYL
A N D R E W D. P U T N A M and T H E R E S A C. P E T E R S O N Department of Medicine, Dalhousie University, Halifax. Nova Scotia. (Canada)
(Received November 3rd, 1990) (Revision received January 24th, 1991)
SUMMARY A measure of the activity of macrophage drug metabolizing enzymes through assay of peripheral monocytes was used to assess the hepatic enzymatic status and thereby evaluate age related changes in drug metabolism. Blood was obtained from elderly subjects (aged 74.8 ± 5.2, ~ 4- S.E., n = 16) and a young control group (aged 23.5 4- 2.0, n = 27). Monocyte A H H activity was used as an index of liver drug metabolism, A L T activity as an index of liver function, monocyte media IL-I and as an index of macrophage activation and serum IL-I levels as a measure of endogenous pyrogenic activity. The medium collected from the cultured monocytes was also assessed for the presence of A H H inhibitory activity. Subjects provided information relating to their age, sex, alcohol consumption, cigarette smoking, recent infection, recent surgery, disease status and medications which could alter drug metabolism. Elderly patients were drawn both from independent seniors living at home and seniors visiting a geriatric day hospital and compared to a control group of young healthy volunteers. Using the experimental design A H H activity did not differ within experimental error between aged (0.832 4- 0.32 nmol/mg prot. per h, n = 16) and young control subjects (0.452 4- 0.17, n = 27). A L T activity did not differ between aged (2.83 I.U. 4- 0.46) and young (4.24 4- 0.82). Monocyte A H H activity did not differ between males (0.45 4- 0.14, n = 33) compared to females (0.65 4- 0.18, n = 29), but was significantly higher in smokers (2.5 4- 1.0, n = 5) compared to nonsmokers (0.35 ± 0.05, n = 52). Mild to moderate alcohol use showed no significant effect on A H H activity. There was no significant difference between the mean level of M C M inhibition of murine hepatocyte A H H between elderly (44.3 ± 8.32°,4,, n = 8) and control (31.5 4- 6.21%, n = 15) subjects, but a larger proportion of the elderCorrespondence to: Dr. T.C. Peterson, Department of Medicine, Clinical Research Centre, Room C103, 5849 University Avenue, Halifax, Nova Scotia, Canada, B3H 4H7.
0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
62 ly population demonstrated such an effect. Serum IL-1 levels (range 0--55.9 pg/ml) were compared to MCM IL-I and A H H inhibitory activity in the elderly and young group.
Key words: Aging; Drug metabolism; Smoking; Arylhydrocarbon hydroxylase; Monocytes INTRODUCTION A significant proportion of the disease states of elderly individuals is related to adverse reactions to drugs [1]. The origins of this high incidence would seem to be multifactorial and complex. Age related changes have been reported for the metabolism of many common drugs but there has been no clear pattern to these observations. Two general trends, however, have been classically reported. The first is that drugs undergoing Phase I reactions, such as oxidation, reduction or hydrolysis are usually metabolized more slowly, while those undergoing conjugation reactions to molecules such as glycine, glucuronic acid, sulfate and glutathione, are usually not influenced by age [2]. Many drugs are metabolized via oxidation (Phase I) by a series of hemoproteins collectively known as cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome bs. Aryi hydrocarbon hydroxylase (AHH) is included among these types of enzymes. This 'mixed function oxidase system' is found in multi-enzymatic form in the smooth endoplasmic reticulum of the hepatocytes and appears in the microsomal fraction of liver homogenate after fractionation of the cells [3]. The cytochrome-P-450-dependent monooygenases are involved in many steps in the metabolism of endogenous compounds, such as steroids, fatty acids, prostaglandins and leukotrienes and also play a key role in the oxidative metabolism of exogenous compounds, such as drugs and other environmental products [4]. Although the contribution of macrophage drug metabolizing enzymes is not large, a measure of their activity through assay of peripheral monocytes has been found to mimic, at least in terms of AHH, the hepatic enzymatic activity status [5,6]. Such a measure is used in this study. Phase II reactions and oxidation of alcohols take place in the soluble cytosolic fraction of liver homogenate and are catalyzed by enzymes that are not part of the mixed function oxidase system and are beyond the scope of this study. In this study, assay of peripheral monocytes was used to further clarify the contribution in activity of the mixed function oxidase system to decreased drug elimination in the elderly. MATERIALS AND METHODS All chemicals used in experimental procedures were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated.
63 Male CFW mice (20--25 g) were obtained from Charles River Farms (Charles River, Ontario) and acclimatized in our own facility for at least 1 week prior to use. Animals were housed on clay chip bedding and fed water and standard Purina mouse chow. Murine hepatocytes were obtained from these animals by the method below for use in A H H inhibition studies. Blood (25 ml) was obtained from elderly subjects (age 65 and over, n = 16) and a young control group (age 18--27, n = 27). This study was approved by the Ethics Committee. Samples of serum were used for alanine aminotransferase (ALT) activity and serum IL-I determinations, isolated monocytes were used for AHH activity determination and monocyte conditioned medium (MCM) (10 ml) was used for MCM IL-1 determination and for A H H inhibition studies involving murine hepatocytes. Monocyte A H H activity was used as an index of liver drug metabolism, ALT activity as an index of liver function, monocyte media IL-I as an index of macrophage activation and serum IL-I levels as a measure of endogenous pyrogenic activity. The media collected from the cultured monocytes was also assessed for the presence of A H H inhibitory activity. The study group subjects were asked to provide information relating to their age, sex, alcohol consumption, cigarette smoking, recent infection, recent surgery, pregnancy (in the young group), disease status and medications such as steroids which could alter drug metabolism. The elderly patient sample population was drawn from elderly patients referred to a geriatric day hospital (n = 10), from independent seniors living at home (n = 6). A group of young healthy volunteers drawn from a university population and the community were used as a control group for comparison with the aged group (n = 27). In studies involving a comparison of the study group to population data, results for the study group were pooled with those for 19 additional healthy volunteers (8 males, 11 females), aged 42 ± 3.0 (-g ± S.E.) to net total population results. This data was then also drawn upon in the assessment of the effect of smoking on A H H activity and in the assessment of the relation between sex and A H H activity. Peripheral blood monocytes were isolated using the method of Peterson [7]. Blood was mixed with RPMI-1640 medium and samples were layered onto Histopaque (Sigma) and centrifuged at 400 x g for 30 min. The opaque interphase was collected and washed with RPMI-1640 at 200 x g for 10 min. Finally, the pellet was resuspended in Leibovitz (L-15) (Sigma) medium supplemented with CPSR-1 (Sigma) and plated and incubated at 37"C for a minimum of 4 h. The medium was then replaced with fresh supplemented L-15 medium. An aliquot of adherent, fixed macrophages was identified cytochemically by the standard method for non-specific esterase staining [7]. The adherent cells were incubated for 24 h at 37"C. After 24 h, the media was collected and filtered through 0.22-#m Millex filters. The macrophage monolayers were then harvested by scraping from culture plates and used for assay of A H H activity. A typical monocyte preparation yielded 6 x 105 monocytes per 10 ml blood. Macrophage A H H activity was determined in cellular homogenates as described
64 previously by the method of Peterson [7], an adaptation of the method of Cantrell and Bresnick [8]. Briefly, cellular homogenates were incubated with benzo[a]pyrene (Sigma) plus cofactor N A D P H (Sigma) at 37"C for 15 min, the reaction was stopped with the addition of acetone and the metabolites of benzo[a]pyrene were extracted with hexane and back extracted into sodium hydroxide. The concentration of 3-hydroxybenzopyrene was determined in the aqueous phase spectrofluorimetrically (Perkin Elmer 30S spectrofluorometer) using excitation 396 nm, emission 522 nm. The fluorescence was compared to 3-hydroxybenzopyrene (3-OHBP) formed per mg cellular protein per h. Cellular protein concentration was determined by the method of Bradford [9]. Murine hepatocytes were isolated by collagenase perfusion as previously described [5]. Briefly, hepatocytes were prepared by retrograde in situ perfusion with 0.05°/~, collagenase (Sigma) using aseptic procedures. Hepatocytes were separated from nonparenchymal cells by gravity settling and differential centrifugation as described in detail previously [5]. The cells were resuspended in Leibovitz (L-15) medium. Cell viability was greater than 85% as determined by trypan blue exclusion. The filtered monocyte conditioned media (MCM) was collected and frozen until assay on hepatocyte preparations. Monocyte conditioned medium (l ml) filtered through 0.22/~m was incubated with hepatocytes (1.5 × l05 cells) in suspension for 2 h at 37"C in a shaking water bath. Hepatocytes were than collected by centrifugation 50 x g 1 min and A H H activity was measured as described above and expressed per mg of cellular protein [9]. Addition of monocyte conditioned medium to hepatocytes without incubation did not alter hepatocyte A H H indicating that monocyte conditioned medium did not neutralize A H H or interfere with its measurement. Addition of monocyte conditioned medium to hepatocytes did not alter hepatocyte viability as determined by trypan blue exclusion. Some factors released from macrophages are temperature sensitive. For example, IL-1 or endogenous pyrogen is sensitive to heating at 70"C for l h [10]. To study heat sensitive factor production, medium obtained as described above was divided and half was heated to 70"C for 1 h prior to incubation with hepatocytes. IL-1 was identified and quantitated in serum and in macrophage media filtrates using an Elisa. The Elisa assay (R&D Systems) for IL-l was chosen because it is l0 times more sensitive than RIA (it detects 4.5 pg of IL-1 per 1-ml sample in serum or buffer) and because it has better reproducibility and is as sensitive as a bioassay. The procedure is carried out in microtitration wells which have been coated with monoclonal antibody specific for IL-1 (solid phase). The color intensity, measured with a microtitration plate reader (~, = 450 nm), is proportional to the amount of bound conjugate and therefore to the amount of IL-I present in the biological sample. ALT activity in serum samples was determined by colorimetric assay using a Sigma ALT kit. An unpaired Student's t-test was used to compare two variables in statistical
65 analyses [l 1]. A level of significance of P < 0.05 was chosen for comparison of sets of data. RESULTS Our results show consistency with population data for the study group (Fig. l) and suggest that aryl hydrocarbon hydroxylase activity demonstrates no statistical change with age (Fig. 2). Increasing sample size of the young population to 46 individuals did not significantly alter the average monocyte A H H activity, though the monocyte A H H activity 0.438 4- 0.10 (n -- 46) demonstrated lower intra group variability due to the larger sample size. Statistical analyses of this larger young control group compared to the elderly group still indicated that there was no significant difference between the young and elderly population with regard to monocyte A H H activity. There was also no statistical difference between aged and young controls for ALT activity (Fig. 3). A H H activity was significantly higher in the smokers compared to non-smokers (Fig. 4). Because our results indicated that smoking had a significant effect on monocyte A H H activity, this enzyme activity was re-examined in the young and the elderly population concentrating only on the non-smoking population to determine if the apparently high values in the elderly population may result from a uneven distribution of smokers in the elderly population. The young non-smoking population had a mean monocyte A H H activity of 0.343 4- 0.036, the non-smoking elderly population had a mean monocyte A H H activity of 0.46 4- 0.18. Statistical analyses of this data indicated that the monocyte A H H activity was not significantly different between the young and elderly non-smoking population. Removal of the smoking population from the elderly group did decrease the mean monocyte A H H activity
0.8
0.0
0,4
AHH Aotlvlty (nmol,/mg. prot,/hr.)
I,
I I
I
I
0.2
I
,,J
8TUDY GROUP
TOTAL POPULATION
n,,48
n-62
Fig.I. AHH activity of the study group (0.58 ± nmol/mg prot. per h (~ ± S.E.)) examined for consistency with population data (0.54 ± 0.11). There was no statistical difference(P > 0.05).
66
1.2
AHH Activity (nmol,/mg. prot./hr,)
0.8 0,8
T
0.4 0.2 0
ELDERLY
OONTROL
n,,'16
n=27
Fig. 2. Effect of age on A H H activity. Both groups demonstrated substantial variability within their group, but there was no statistical difference (P ~- 0.05) between the elderly (0.832 + 0.32 nmol/mg prot. per h) and young control subjects (0.452 ~ 0.17) in the activity of this mixed function oxidase.
from 0.83 to 0.46 suggesting that smoking was a significant factor leading to high levels observed in the elderly population. Mild to moderate alcohol use had no significant effect on A H H activity in our study (data not shown). No statistical difference was found in A H H activity between males and females in either the young or old group. A H H did not change in either the males or females with respect to age (Table I).
O
ALT Aotlvlty (I.U./rag. prot.)
t ELDERLY
I ~NTROL
N=IB Fig. 3. Effect of age on A L T activity. There was no statistical difference between aged (2.83 I.U. 4- 0.46) and young control (4.24 + 0.82) subjects (P ~, 0.05).
67
4
AHH Aotlvlty (nmol./mg, prot./hr.)
8.6
I
0 2.5 2 1.6 1
0,5!
I
0
=
I
NON-SMOKER8
SMOKER8
Fig. 4. Influence of smokin 8 on AHH activity. Although the number of smokers was relatively small, they were found to have a markedly elevated (P ~, 0.05) AHH activity (2.5 ± 1.0 nmol/mg prot. per h) when compared to non-smokers (0.35 4- 0.05).
In studies involving the effect of the monocyte conditioned media on murine hepatocytes, a heat sensitive inhibitory effect was found in representative groups of both young and elderly subjects. There was no significant difference in the level of inhibition between the young and elderly subjects which displayed the effect (Fig. 5). A smaller proportion of the control group, however, demonstrated an MCM inhibitory effect as compared with the elderly leading to an overall mean inhibitory effect which was statistically greater than the elderly for the representative group. Interleukin-1 was measured in serum from elderly and young patients and ranged from 0 to 55.9 pg/ml. No difference was observed between the two groups. Measurement of IL-1 in MCM indicated that no difference was observed between the elderly group (79 + 19 pg/ml) and the young group (120 + 35 pg/ml).
TABLE I COMPARISON OF MEAN AHH ACTIVITY IN Y O U N G A N D OLD MALES A N D FEMALES A N D AN EVALUATION FOR A SEX SPECIFIC ALTERATION IN AVERAGE AHH ACTIVITY WITH A G I N G Mean A H H Activity (nmol/mg prot. per h)
Elderly Controls Total
Male
n
Female
n
0.36 4- 0.05 0.48 4- 0.19 0.46 4- 0.14
6 27 33
1.12 4- 0.50 0.40 4- 0.06 0.65 ± 0.18
10 19 29
Results are expressed as 2 ± S.E. for the number of individuals shown.
68 AHH INHIBITION (%) 00 50 4.0
I
30 20 10 O
ELDERLY
OONTROL
n,,8
11~,'16
Fig. 5. Comparison of the percentage AHH inhibition of untreated versus heat treated (70°C for 1 h) MCM in aged and control subjects. There was no significant difference between the mean level of MCM inhibition of murine hepatocyte AHH between elderly (44.3 + 3.32%) and control (31.5 + 6.21%) subjects, but a larger proportion (n = 8 of 8) of the elderly population demonstrated such an effect as compared to the controls (n = 15 of 33) in the randomly selected representatives.
DISCUSSION O u r results show no statistical change in m o n o c y t e A H H activity with aging and hence support the data o f primate and in vivo h u m a n microsomal studies which contend that a decline in the specific activities o f the h u m a n monooxygenases does not account for the decline in the systemic clearance o f drugs undergoing hepatic oxidation. T h o u g h the aging population was not statistically different from the y o u n g population with respect to m o n o c y t e A H H activity our results did show that the m o n o c y t e A H H activity in the elderly showed a tendency to be higher. In addition both groups showed high intra g r o u p variability for both A H H and A L T , i.e. high degree o f overlap was observed between the elderly and the y o u n g groups. In order to confirm our initial observations, the sample sizes were increased. Analyses o f the larger sized sample groups o f y o u n g and elderly subjects again indicated that there was no statistical difference between the m o n o c y t e A H H activity in elderly and y o u n g subjects within experimental error using this experimental design. A L T activity was normal in both y o u n g and elderly population indicating no underlying liver dysfunction. The results also indicate that no significant statistical difference was observed for A L T between the y o u n g and elderly population within experimental error using this experimental design. As noted t h o u g h high intra g r o u p variability was observed for A L T in both the y o u n g and elderly population. A H H activity was~ however, clearly found to be statistically higher in smokers as c o m p a r e d to nonsmokers, supporting earlier conclusions drawn from drug model studies that drug
69 metabolizing enzymes are induced with smoking. In order to determine if smoking was affecting the monocyte A H H results in the aging population more so than in the young population the monocyte A H H activity was then statistically analyzed in elderly and young non-smoking populations. As indicated in the results section removal of this smoking population from both the young and elderly group lowered the mean monocyte AHH activity in both groups. The affect on the elderly population appeared to be greater; the mean monocyte AHH activity in the elderly group was reduced from 0.83 to 0.46 with removal of the non-smokers. Statistical analysis of the non-smoking young and elderly group again clearly indicated that there was no significant difference in monocyte AHH activity between the young and elderly non-smoking population within experimental error using this experimental design. Also removal of the non-smoking population from the total group significantly reduced the intra-group variability. In the elderly, several age related changes in anatomy and physiology have been found to correlate with alterations in hepatic metabolism of drugs in this population. These things include a reduction in liver size, a decrease in liver blood flow and an alteration in synthesis of drug binding proteins [3,12]. Wynne et al. [13] found a significant reduction in liver volume, apparent liver blood flow and perfusion and suggest this may at least partially account for the decline in the clearance of many drugs undergoing liver metabolism, particularly those of high extraction. The clearance of capacity limited drugs is influenced by the extent of plasma protein binding and, in general, those drugs (acidic) which bind to albumin tend to exhibit an increased free fraction, while those binding to a-acid glycoprotein tend to exhibit a decreased free fraction with age due to a decrease and increase, respectively, in these serum proteins [14]. This trend, however, has many exceptions and Vestal [15] suggests the clinical significance of age related changes is small and that other factors contribute in larger degree to inter-individual variability in protein binding. It has also been implied that age related changes occur due to an impairment in the activity of drug metabolizing enzymes. In the rat, particularly in the male, it has been well documented that the specific activities of a variety of hepatic microsomal monooxygenases fall with age both in relation to the content of microsomal protein and liver weight [16]. Recently Mote et al. [17] investigated the influence of age on liver gene expression and found a progressive decrease in basal Pi and P3-450 RNA levels between 4 and 28 months of age in BI0.RIII mice, but not C57.BL/10 mice. By using an antibody against the major 'male specific' species of cytochrome P-450, Fujita et al. [18] have provided evidence that differences in the amount of this isoenzyme (P-450ml) may be responsible for the age related and sex associated differences in some of the drug metabolizing activities in rats. He further proposes [18] that senescence associated feminization of the drug metabolizing ability of the male rat liver may be due in part to the decrease in testosterone levels in old age. Taken together with the work of Mote et al. [17] on age related changes in P-450 gene expression, an elegant animal model emerges for the senescence associated [9]change
70 of the relative abundance o f multiple species of P-450 in the liver into the female pattern. Caution is urged in the extrapolation of these changes to humans since the alterations in enzyme activity were species, strain, sex and substrate specific. This model, in addition, has not been corroborated by primate (Macacu monkey) models where no significant age related change was detected in the mixed function oxidase enzymes studied [19,201 nor has it been corroborated in humans [21,22]. In humans there is limited data on in vitro drug metabolizing enzyme capacity, while data from in vivo studies involving the mixed function oxidase system has been contradictory. In vivo studies using antipyrine as the model substrate have demonstrated an age associated reduction in the metabolic clearance for this drug [23]. Since antipyrine is a low extraction drug and is minimally bound to plasma proteins, its systemic clearance approximates drug metabolizing enzyme activity. It has hence been suggested that the age dependent decline in antipyrine clearance is indicative of a decline of the activities of the microsomal mixed function oxidase enzyme with age. Recent research [23], however, finds no evidence for a decrease in hepatic microsomal function with advancing age as measured by the aminopyrine breath test (n = 60). In addition, Van Gell and Van Bezooijen [24] found no changes with aging in the partial clearances of the antipyrine metabolites 3-hydroxymethylantipyrine (HMA) and 4-hydroxyantipyrine (OHA) in vivo, but a decrease from age 3 to 24 months followed by an increase up to age 36 months for norantipyrine (NORA) in rats. Thus, the collective data for age related changes in the mixed function oxidase system predicted by this model would seem ambiguous at best. In a study in which actual tissue samples were obtained at cholecystectomy or vagotomy from 17 patients, no correlation was observed between age and microsomal protein recovery, maximum enzyme activity or apparent enzyme activity [12]. Since this involved a select group of elderly patients fit enough for surgery, fears have been expressed about whether such findings can be generalized, especially with regard to the frail old. Our results for human male versus female A H H corroborate our earlier suggestion [6] that there is no sex related difference in human monocyte A H H activity nor is there any correlation between decreasing A H H activity and increasing age in these groups. As a result, our data for A H H activity as a whole suggests that the rodent models [17,18] of selective isozyme changes with aging may not be applicable to humans. In this study, MCM from both young and elderly subjects was shown to inhibit murine hepatocyte A H H , when compared to heat treated M C M samples, suggesting that release of a heat sensitive factor may be altering the A H H activity in certain individuals and may be contributing to population variability in A H H activity. One factor which may be acting to produce this effect is IL-1. 1L-1 has roles in immunity, inflammation and connective tissue metabolism and is made up of two subtypes, alpha and beta, with a common receptor on target cells [25]. In a study to determine the cause of endotoxin mediated depression of P-450
71 levels, Shedlofsky [26] showed significant depression of P-450 levels by injection of IL-1 into mice. Sujita [27] has found that cultured rat hepatocytes express a considerable amount of IL-1 receptor and that incubation of these hepatocytes with I L-1 for 18 h decreased the contents of P-450 in a dose dependent manner. Our results [28] have found that in the presence of antibody to IL-I, the inhibitory effect of MCM media on hepatocyte AHH activity is significantly reduced. It is interesting to note in this regard that Yamaguchi et al. [29] have found that the amount of IL-I released from LPS-stimulated alveolar macrophages was significantly decreased in smokers compared with that in non-smokers. Such a factor may be contributing to the markedly elevated monocyte AHH activity we have reported for smokers. Based on this evidence, then, there seems to be emerging a concept of a modulatory role of IL-I on drug metabolizing activity. In freshly isolated monocytes or macrophages derived from monocytes in vitro, Lewis et al. [30] have found a size dependent functional heterogeneity in the amount of IL-1 released per cell and in the number of cells secreting detectable levels of these products at one particular time. The different fraction of elderly subjects exhibiting a MCM inhibitory effect, as compared with young subjects, could indicate some sort of alteration in this heterogeneity with aging. Despite its lack of a direct triggering action, Cavaillon et al. [31] have found that human recombinant C5a is able to act synergistically with LPS, leading to higher TNF and IL-I release-by human monocytes. They propose that enhanced release of these two factors might occur during Gram-negative infections, since LPS is known to lead to C activation. Tatsuno et al. [ 32] found, moreover, a significant augmentation of IL-l-like activity in monocytes by leukotriene B4, while Laurenzii et al. [33] reported a similar effect for substance P. Taken together, these findings suggest there could be a number of intermediary factors modulating a possible alteration in the heterogeneity of IL-1 release with aging. As pointed out by Bradley et al. [34] it has long been postulated that deficiencies in the production of certain monokines could account for the diminished febrile responses and increased severity of infections in the elderly. They found, however, that while in vitro assays appeared to show a decrease in IL-I and TNF activity, endogenous pyrogen assays showed normal production of IL-I and TNF by macrophages of aged rats. One might speculate that it may be intermediary factors, like those referred to above, acting to trigger IL-I release in vivo, but not in vitro, which could be influencing blood monocytes like the macrophages in this study. Recently, Arend et al. [35] have further found that human monocytes cultured on adherent IgG produce a specific IL-I inhibitor that functions as a receptor antagonist (IL-ira) and suggest an important role for this substance in normal and pathophysiologicai states. This would seem to provide another alternative explanation for the decreased secretion of IL-I in aged macrophages found by Bradley et al. [34] in vitro and possibly reveals a further modulatory factor which may be deranged in aging. We have also found [28] that catalase, which inactivates activated oxygen species,
72
also reduces a component of the inhibitory effect of the MCM on hepatocyte AHH. Interestingly enough, a study by Ischiropoulos et al. [36] indicates that in rats, the proportion of the relatively inactive forms of Cu, Zn superoxide dismutase increase with age, while Niwa et al. [37] have shown that levels of lipid peroxides in elderly humans may be elevated as compared to the population in general (P < 0.05). Thus, altered activated oxygen disposition in aging may have an important role relating to drug metabolism. Azri and Renton [38] have speculated that the inhibition of P-450 involved in Listeria infections may be caused by the hemolysin produced by this organism acting to damage the mixed function oxidase system. Renton and Knickle [39] have shown that interferon inducers depress the m R N A for the P-450LA isoenzyme and suggest a role for interferon mediated P-450 depression in viral infections. Hence, a complex network of factors, whose specific mode of interaction remains to be worked out, may be operating to produce the inhibitory effect observed in our studies. The fact that the elderly population had a larger fraction of subjects with an MCM inhibitory effect suggests that the interactions in this group may differ as compared with young controls, at least as pertains to the subjects in this study. In conclusion we have shown: (1) mean monocyte A H H activity does not show a statistically significant change with aging; (2) A H H activity is statistically significantly higher for smokers; (3) no statistical difference exists in monocyte A H H activity between young or old males and females; (4) the existence of an inhibitory effect of MCM media on hepatocyte AHH, present in similar degree, but with a greater prevalence, in the elderly as compared to the control population. A C K N O W L E D G E M ENTS
Thanks to Richard A. Isbrucker for his invaluable technical assistance, Dr. David Hogan for his aid in securing the geriatric population, the Dalhousie Medical Research Foundation for studentship support (A.D.P.) and Lorna Russell for secretarial assistance. This study was supported by the Medical Research Council of Canada and the Dalhousie Medical Research Foundation. REFERENCES 1 2 3
R.G. McAllister, Age-related changes in drug handling in man, A Symposium: Lipids and Hypertension in the Elderly. M.D. Rawlins, O.F.W. James, F.M. Williams, H. Wynne and K.W. Woodhouse, Age and the metabolism of drugs. Q. J. Meal, 545 (1987) 64~243. C. Loi and R.E. Vestal, Drug metabolism in the elderly. Pharmacol. Ther., 36 (1988) 131.
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D. Mansuy, P. Battioni and J.P. Battioni, Chemical model systems for drug-metabolizing cytochrome-P-450---dependent monooxygenases. Eur. J. Biochem., 184 (1989) 267. T.C. Peterson and K.W. Renton, Depression of cytochrome P-450-dependent drug biotransformation in hepatocytes after the activation of the reticuloendothelial system by dextran sulfate. J. Pharm. Exp. Ther., 299 (1984) 221--229. T.C. Peterson and C.N. Williams, Depression of peripheral blood monocyte aryl hydrocarbon hydroxylase activity inpatients with liver disease: possible involvement of macrophage factors. Hepatology, 333 (1987) 2--7. T.C. Peterson, Drug-metabolizing enzymes in rat, mouse, pig and human macrophages and the effect of phagocytic activation. 3911 (1987) 22--36. E. Cantrell and E. Bresnick, Benzopyrene hydroxylase activity in isolated parenchymal and nonparenchymal cells of rat liver. J. Cell. Biol., 52 (1972) 316. M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 72 (1976) 248. F. Haesler, P. Bodel and E. Atkins, Characteristics of pyrogen production by isolated rabbit Kupffer cells in vitro. J. Reticuloendothelial Soc., 569 (1977) 6--22. J.H. Zar, Biostatistical Methods, Prentice-Hall, N.J., 1974. K.W. Woodhouse and H.A. Wynee, Age-related changes in liver size and hepatic blood flow, the influence on drug metabolism in the elderly. 15 (1988) 287. H.A. Wynne, L.H. Cope, E. Mutch, M.D. Rawlins, K.W. Woodhouse and O.F.W. James, The effect of age upon liver volume and apparent liver blood flow in healthy man. 297 (1989) 2--9. S.M. Wallace and R.K. Verbeeck, Plasma protein binding of drugs in the elderly. Clin. Pharmacokinet., 12 (1987) 41. R.E. Vestal, Aging and determinants of hepatic drug clearance, Hepatology. 331 (1989) 2--9. D.L. Schmucker, D.A. Vassey, R.K. Wang, J.L. James and A. Maloney, Age-dependent alterations in the physicochemical properties of rat liver microsomes. Mech. Ageing Dev., 27 (1984) 207--217. P.L. Mote, J.M. Grizzle, R.L. Walford and S.R. Spindler, Age related down regulation of hepatic cytochrome Pi-450, P3-450, catalase and CuZn-superoxide dismutase RNA. Mech. Ageing Dev.. 53 (1990) 101--110. S. Fujita, M. Chiba, M. Ohta, K. Kitani and T. Suzuki, Alternation of plasma sex hormone levels associated with old age and its effect on hepatic drug metabolism in rats. J. Pharm. Exp. Ther., 369 (1990) 251--253. M.A. Sutter, W.G. Wood, L.S. Williamson, R. Strong, K. Pickham and A. Richardson, Comparison of the hepatic mixed function oxidase system of young, adult and old non-human primates (Macaca nemestrina). Biochem. Pharm., 2983 (1985) 16~34. A.G. Maloney, D.L. Schmucker, D.S. Vessey and R.K. Wang, The effects of aging on the hepatic microsomat mixed-function oxidase system of male and female monkeys. Hepatology, 282 (1986) 2--6. K.W. Woodhouse, E. Mutch, F.M. Williams, M.D. Rawlings, and O.F.W. James, The effect of age on pathways of drug metabolism in human liver. Age and Ageing, 13 (1984) 328. R.E. Vestal, E.A. McGuire, J.D. Tobin, B.H. Cohen, N.W. Schock, and R. Andres, Antipyrine metabolism in man: influence of age, alcohol, caffeine and smoking. Clin. Pharmacol. Ther., 18 (1975) 425. S. Arora, Z. Kassarjian, S.D. Krasinski, B. Croffey, M.M. Kaplan and R.M. Russell, Effect ofage on tests of intestinal and hepatic function in healthy humans. Gastroenterology, 96 (1989) 1560. C.A.J.F. Van Gell and C.F.A. Van Bezooijen, The effect of age on the oxidative metabolism of antipyrine by uninduced microsomal fractions of rat liver. Mech. Ageing Dev., 53 (1990) 169--177. J.C. Manson, J.A. Symons, F.S. DiGiovine, S. Poole and G.W. Duff, Autoregulation of interleukin 1 production. Eur. J. lmrnunol., 19 (1989) 271. S.I. Shedlofsky, A.T. Swim, J.M. Robinson, V.S. Callicchio, D.A. Cohen and C.J. McClain, Interleukin-I (IL-I) depresses cytochrome P-450 levels and activities in mice. Life Sci.. 40 (1987) 2331. K. Sujita, F. Okuno, Y. Tanaka, Y. Hirano, Y. Inamoto, S. Eto and A. Masao, Effect of interleukin 1 (1L 1) on the levels of cytochrome P-450 involving I L-1 receptor on the isolated hepatocytes of rat. 1217 (1990) 163--168.
74 28 29 30
31
32
33
34 35 36 37
38 39
T.C. Peterson, lnterleukin-I and free radicals in liver disease. Cytokine, 1 (1989) 140, 407. E. Yamaguchi, N. Okazaki, A. itoh, S. Abe, Y. Kawakami and H. Okuyama, Interleukin 1 Production by alveolar macrophages is decreased in smokers. Am. Rev. Respir. Dis., 140 (1989) 397. C.E. Lewis, S.P. McCarthy, J. Lorenzen and J.O'D. McGee, Heterogeneity among human mononuclear phagocytes in their secretion of lysozyme, interleukin 1 and type-transforming growth fact: a quantitative analysis at the single-cell level. Eur. J. Irnmunol., 19 (1989) 2037. J.M. Cavaillon, C. Fitting and N. Haeffner-Cavaillon, Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur. J. lmmunol., 20 (1990) 253. I. Tatsuno, H. Saito, K.J. Chang, Y. Tamura and S. Yoshida, Comparison of the effect between leukotriene B4 and leukotriene B5 on the induction of interleukin l-like activity and calcium mobilizing activity in human blood monocytes. Agents and Actions, 29 (1990) 3. M.A. Laurenzi, A.A. Persson, C.J. Dalsgaard and A. Hegerstrand, The neuropeptide substance P stimulates production of interleukin 1 in human blood monocytes: Activated cells are preferentially influenced by the neuropeptide. Scand. J. lmmunol., 31 (1990) 259. S.F. Bradley, A. Vibhagool, S.L. Kunkel and C.A. Kauffman, Monokine secretion in aging and protein malnutrition. J. Leukocyte Biol., 45 (1989) 510. W.P. Arend, H.G. Welgus, R.C. Thompson and S.P. Eisenberg, Biological properties of recombinant human monocyte-derived interleukin 1 receptor antagonist. J. Clin. Invest.. 85 (1990) 1694. H. Ischiropoulos, C.E. Nadziejko and Y. Kikkawa, Effect of aging on pulmonary superoxide dismutase. Mech. Ageing Dev., 52 (1990) l I 26. Y. Niwa, T. Kasama, Y. Miyachi and T. Kanoh, Neutrophil chemotaxis, phagocytosis and parameters of reactive oxygen species in human aging: cross-sectional and longitudinal studies. L!'[i, Sci., 44 (1989) 1655. S. Azri and K.W. Renton, Depression of murine hepatic mixed function oxidase during infection with Listeria monoeytogenes. J. Pharrn. Exp. Ther., 1089 (1987) 233--243. K.W. Renton and L.C. Knickle, Regulation of hepatic cytochrome P-450 during infectious disease. Can. J. PhysioI.Pharmaeol., 68 (1989) 777.
61
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF A G I N G AND O T H E R F A C T O R S ON M O N O C Y T E H Y D R O C A R B O N H Y D R O X Y L A S E ACTIVITY
ARYL
A N D R E W D. P U T N A M and T H E R E S A C. P E T E R S O N Department of Medicine, Dalhousie University, Halifax. Nova Scotia. (Canada)
(Received November 3rd, 1990) (Revision received January 24th, 1991)
SUMMARY A measure of the activity of macrophage drug metabolizing enzymes through assay of peripheral monocytes was used to assess the hepatic enzymatic status and thereby evaluate age related changes in drug metabolism. Blood was obtained from elderly subjects (aged 74.8 ± 5.2, ~ 4- S.E., n = 16) and a young control group (aged 23.5 4- 2.0, n = 27). Monocyte A H H activity was used as an index of liver drug metabolism, A L T activity as an index of liver function, monocyte media IL-I and as an index of macrophage activation and serum IL-I levels as a measure of endogenous pyrogenic activity. The medium collected from the cultured monocytes was also assessed for the presence of A H H inhibitory activity. Subjects provided information relating to their age, sex, alcohol consumption, cigarette smoking, recent infection, recent surgery, disease status and medications which could alter drug metabolism. Elderly patients were drawn both from independent seniors living at home and seniors visiting a geriatric day hospital and compared to a control group of young healthy volunteers. Using the experimental design A H H activity did not differ within experimental error between aged (0.832 4- 0.32 nmol/mg prot. per h, n = 16) and young control subjects (0.452 4- 0.17, n = 27). A L T activity did not differ between aged (2.83 I.U. 4- 0.46) and young (4.24 4- 0.82). Monocyte A H H activity did not differ between males (0.45 4- 0.14, n = 33) compared to females (0.65 4- 0.18, n = 29), but was significantly higher in smokers (2.5 4- 1.0, n = 5) compared to nonsmokers (0.35 ± 0.05, n = 52). Mild to moderate alcohol use showed no significant effect on A H H activity. There was no significant difference between the mean level of M C M inhibition of murine hepatocyte A H H between elderly (44.3 ± 8.32°,4,, n = 8) and control (31.5 4- 6.21%, n = 15) subjects, but a larger proportion of the elderCorrespondence to: Dr. T.C. Peterson, Department of Medicine, Clinical Research Centre, Room C103, 5849 University Avenue, Halifax, Nova Scotia, Canada, B3H 4H7.
0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
62 ly population demonstrated such an effect. Serum IL-1 levels (range 0--55.9 pg/ml) were compared to MCM IL-I and A H H inhibitory activity in the elderly and young group.
Key words: Aging; Drug metabolism; Smoking; Arylhydrocarbon hydroxylase; Monocytes INTRODUCTION A significant proportion of the disease states of elderly individuals is related to adverse reactions to drugs [1]. The origins of this high incidence would seem to be multifactorial and complex. Age related changes have been reported for the metabolism of many common drugs but there has been no clear pattern to these observations. Two general trends, however, have been classically reported. The first is that drugs undergoing Phase I reactions, such as oxidation, reduction or hydrolysis are usually metabolized more slowly, while those undergoing conjugation reactions to molecules such as glycine, glucuronic acid, sulfate and glutathione, are usually not influenced by age [2]. Many drugs are metabolized via oxidation (Phase I) by a series of hemoproteins collectively known as cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome bs. Aryi hydrocarbon hydroxylase (AHH) is included among these types of enzymes. This 'mixed function oxidase system' is found in multi-enzymatic form in the smooth endoplasmic reticulum of the hepatocytes and appears in the microsomal fraction of liver homogenate after fractionation of the cells [3]. The cytochrome-P-450-dependent monooygenases are involved in many steps in the metabolism of endogenous compounds, such as steroids, fatty acids, prostaglandins and leukotrienes and also play a key role in the oxidative metabolism of exogenous compounds, such as drugs and other environmental products [4]. Although the contribution of macrophage drug metabolizing enzymes is not large, a measure of their activity through assay of peripheral monocytes has been found to mimic, at least in terms of AHH, the hepatic enzymatic activity status [5,6]. Such a measure is used in this study. Phase II reactions and oxidation of alcohols take place in the soluble cytosolic fraction of liver homogenate and are catalyzed by enzymes that are not part of the mixed function oxidase system and are beyond the scope of this study. In this study, assay of peripheral monocytes was used to further clarify the contribution in activity of the mixed function oxidase system to decreased drug elimination in the elderly. MATERIALS AND METHODS All chemicals used in experimental procedures were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated.
63 Male CFW mice (20--25 g) were obtained from Charles River Farms (Charles River, Ontario) and acclimatized in our own facility for at least 1 week prior to use. Animals were housed on clay chip bedding and fed water and standard Purina mouse chow. Murine hepatocytes were obtained from these animals by the method below for use in A H H inhibition studies. Blood (25 ml) was obtained from elderly subjects (age 65 and over, n = 16) and a young control group (age 18--27, n = 27). This study was approved by the Ethics Committee. Samples of serum were used for alanine aminotransferase (ALT) activity and serum IL-I determinations, isolated monocytes were used for AHH activity determination and monocyte conditioned medium (MCM) (10 ml) was used for MCM IL-1 determination and for A H H inhibition studies involving murine hepatocytes. Monocyte A H H activity was used as an index of liver drug metabolism, ALT activity as an index of liver function, monocyte media IL-I as an index of macrophage activation and serum IL-I levels as a measure of endogenous pyrogenic activity. The media collected from the cultured monocytes was also assessed for the presence of A H H inhibitory activity. The study group subjects were asked to provide information relating to their age, sex, alcohol consumption, cigarette smoking, recent infection, recent surgery, pregnancy (in the young group), disease status and medications such as steroids which could alter drug metabolism. The elderly patient sample population was drawn from elderly patients referred to a geriatric day hospital (n = 10), from independent seniors living at home (n = 6). A group of young healthy volunteers drawn from a university population and the community were used as a control group for comparison with the aged group (n = 27). In studies involving a comparison of the study group to population data, results for the study group were pooled with those for 19 additional healthy volunteers (8 males, 11 females), aged 42 ± 3.0 (-g ± S.E.) to net total population results. This data was then also drawn upon in the assessment of the effect of smoking on A H H activity and in the assessment of the relation between sex and A H H activity. Peripheral blood monocytes were isolated using the method of Peterson [7]. Blood was mixed with RPMI-1640 medium and samples were layered onto Histopaque (Sigma) and centrifuged at 400 x g for 30 min. The opaque interphase was collected and washed with RPMI-1640 at 200 x g for 10 min. Finally, the pellet was resuspended in Leibovitz (L-15) (Sigma) medium supplemented with CPSR-1 (Sigma) and plated and incubated at 37"C for a minimum of 4 h. The medium was then replaced with fresh supplemented L-15 medium. An aliquot of adherent, fixed macrophages was identified cytochemically by the standard method for non-specific esterase staining [7]. The adherent cells were incubated for 24 h at 37"C. After 24 h, the media was collected and filtered through 0.22-#m Millex filters. The macrophage monolayers were then harvested by scraping from culture plates and used for assay of A H H activity. A typical monocyte preparation yielded 6 x 105 monocytes per 10 ml blood. Macrophage A H H activity was determined in cellular homogenates as described
64 previously by the method of Peterson [7], an adaptation of the method of Cantrell and Bresnick [8]. Briefly, cellular homogenates were incubated with benzo[a]pyrene (Sigma) plus cofactor N A D P H (Sigma) at 37"C for 15 min, the reaction was stopped with the addition of acetone and the metabolites of benzo[a]pyrene were extracted with hexane and back extracted into sodium hydroxide. The concentration of 3-hydroxybenzopyrene was determined in the aqueous phase spectrofluorimetrically (Perkin Elmer 30S spectrofluorometer) using excitation 396 nm, emission 522 nm. The fluorescence was compared to 3-hydroxybenzopyrene (3-OHBP) formed per mg cellular protein per h. Cellular protein concentration was determined by the method of Bradford [9]. Murine hepatocytes were isolated by collagenase perfusion as previously described [5]. Briefly, hepatocytes were prepared by retrograde in situ perfusion with 0.05°/~, collagenase (Sigma) using aseptic procedures. Hepatocytes were separated from nonparenchymal cells by gravity settling and differential centrifugation as described in detail previously [5]. The cells were resuspended in Leibovitz (L-15) medium. Cell viability was greater than 85% as determined by trypan blue exclusion. The filtered monocyte conditioned media (MCM) was collected and frozen until assay on hepatocyte preparations. Monocyte conditioned medium (l ml) filtered through 0.22/~m was incubated with hepatocytes (1.5 × l05 cells) in suspension for 2 h at 37"C in a shaking water bath. Hepatocytes were than collected by centrifugation 50 x g 1 min and A H H activity was measured as described above and expressed per mg of cellular protein [9]. Addition of monocyte conditioned medium to hepatocytes without incubation did not alter hepatocyte A H H indicating that monocyte conditioned medium did not neutralize A H H or interfere with its measurement. Addition of monocyte conditioned medium to hepatocytes did not alter hepatocyte viability as determined by trypan blue exclusion. Some factors released from macrophages are temperature sensitive. For example, IL-1 or endogenous pyrogen is sensitive to heating at 70"C for l h [10]. To study heat sensitive factor production, medium obtained as described above was divided and half was heated to 70"C for 1 h prior to incubation with hepatocytes. IL-1 was identified and quantitated in serum and in macrophage media filtrates using an Elisa. The Elisa assay (R&D Systems) for IL-l was chosen because it is l0 times more sensitive than RIA (it detects 4.5 pg of IL-1 per 1-ml sample in serum or buffer) and because it has better reproducibility and is as sensitive as a bioassay. The procedure is carried out in microtitration wells which have been coated with monoclonal antibody specific for IL-1 (solid phase). The color intensity, measured with a microtitration plate reader (~, = 450 nm), is proportional to the amount of bound conjugate and therefore to the amount of IL-I present in the biological sample. ALT activity in serum samples was determined by colorimetric assay using a Sigma ALT kit. An unpaired Student's t-test was used to compare two variables in statistical
65 analyses [l 1]. A level of significance of P < 0.05 was chosen for comparison of sets of data. RESULTS Our results show consistency with population data for the study group (Fig. l) and suggest that aryl hydrocarbon hydroxylase activity demonstrates no statistical change with age (Fig. 2). Increasing sample size of the young population to 46 individuals did not significantly alter the average monocyte A H H activity, though the monocyte A H H activity 0.438 4- 0.10 (n -- 46) demonstrated lower intra group variability due to the larger sample size. Statistical analyses of this larger young control group compared to the elderly group still indicated that there was no significant difference between the young and elderly population with regard to monocyte A H H activity. There was also no statistical difference between aged and young controls for ALT activity (Fig. 3). A H H activity was significantly higher in the smokers compared to non-smokers (Fig. 4). Because our results indicated that smoking had a significant effect on monocyte A H H activity, this enzyme activity was re-examined in the young and the elderly population concentrating only on the non-smoking population to determine if the apparently high values in the elderly population may result from a uneven distribution of smokers in the elderly population. The young non-smoking population had a mean monocyte A H H activity of 0.343 4- 0.036, the non-smoking elderly population had a mean monocyte A H H activity of 0.46 4- 0.18. Statistical analyses of this data indicated that the monocyte A H H activity was not significantly different between the young and elderly non-smoking population. Removal of the smoking population from the elderly group did decrease the mean monocyte A H H activity
0.8
0.0
0,4
AHH Aotlvlty (nmol,/mg. prot,/hr.)
I,
I I
I
I
0.2
I
,,J
8TUDY GROUP
TOTAL POPULATION
n,,48
n-62
Fig.I. AHH activity of the study group (0.58 ± nmol/mg prot. per h (~ ± S.E.)) examined for consistency with population data (0.54 ± 0.11). There was no statistical difference(P > 0.05).
66
1.2
AHH Activity (nmol,/mg. prot./hr,)
0.8 0,8
T
0.4 0.2 0
ELDERLY
OONTROL
n,,'16
n=27
Fig. 2. Effect of age on A H H activity. Both groups demonstrated substantial variability within their group, but there was no statistical difference (P ~- 0.05) between the elderly (0.832 + 0.32 nmol/mg prot. per h) and young control subjects (0.452 ~ 0.17) in the activity of this mixed function oxidase.
from 0.83 to 0.46 suggesting that smoking was a significant factor leading to high levels observed in the elderly population. Mild to moderate alcohol use had no significant effect on A H H activity in our study (data not shown). No statistical difference was found in A H H activity between males and females in either the young or old group. A H H did not change in either the males or females with respect to age (Table I).
O
ALT Aotlvlty (I.U./rag. prot.)
t ELDERLY
I ~NTROL
N=IB Fig. 3. Effect of age on A L T activity. There was no statistical difference between aged (2.83 I.U. 4- 0.46) and young control (4.24 + 0.82) subjects (P ~, 0.05).
67
4
AHH Aotlvlty (nmol./mg, prot./hr.)
8.6
I
0 2.5 2 1.6 1
0,5!
I
0
=
I
NON-SMOKER8
SMOKER8
Fig. 4. Influence of smokin 8 on AHH activity. Although the number of smokers was relatively small, they were found to have a markedly elevated (P ~, 0.05) AHH activity (2.5 ± 1.0 nmol/mg prot. per h) when compared to non-smokers (0.35 4- 0.05).
In studies involving the effect of the monocyte conditioned media on murine hepatocytes, a heat sensitive inhibitory effect was found in representative groups of both young and elderly subjects. There was no significant difference in the level of inhibition between the young and elderly subjects which displayed the effect (Fig. 5). A smaller proportion of the control group, however, demonstrated an MCM inhibitory effect as compared with the elderly leading to an overall mean inhibitory effect which was statistically greater than the elderly for the representative group. Interleukin-1 was measured in serum from elderly and young patients and ranged from 0 to 55.9 pg/ml. No difference was observed between the two groups. Measurement of IL-1 in MCM indicated that no difference was observed between the elderly group (79 + 19 pg/ml) and the young group (120 + 35 pg/ml).
TABLE I COMPARISON OF MEAN AHH ACTIVITY IN Y O U N G A N D OLD MALES A N D FEMALES A N D AN EVALUATION FOR A SEX SPECIFIC ALTERATION IN AVERAGE AHH ACTIVITY WITH A G I N G Mean A H H Activity (nmol/mg prot. per h)
Elderly Controls Total
Male
n
Female
n
0.36 4- 0.05 0.48 4- 0.19 0.46 4- 0.14
6 27 33
1.12 4- 0.50 0.40 4- 0.06 0.65 ± 0.18
10 19 29
Results are expressed as 2 ± S.E. for the number of individuals shown.
68 AHH INHIBITION (%) 00 50 4.0
I
30 20 10 O
ELDERLY
OONTROL
n,,8
11~,'16
Fig. 5. Comparison of the percentage AHH inhibition of untreated versus heat treated (70°C for 1 h) MCM in aged and control subjects. There was no significant difference between the mean level of MCM inhibition of murine hepatocyte AHH between elderly (44.3 + 3.32%) and control (31.5 + 6.21%) subjects, but a larger proportion (n = 8 of 8) of the elderly population demonstrated such an effect as compared to the controls (n = 15 of 33) in the randomly selected representatives.
DISCUSSION O u r results show no statistical change in m o n o c y t e A H H activity with aging and hence support the data o f primate and in vivo h u m a n microsomal studies which contend that a decline in the specific activities o f the h u m a n monooxygenases does not account for the decline in the systemic clearance o f drugs undergoing hepatic oxidation. T h o u g h the aging population was not statistically different from the y o u n g population with respect to m o n o c y t e A H H activity our results did show that the m o n o c y t e A H H activity in the elderly showed a tendency to be higher. In addition both groups showed high intra g r o u p variability for both A H H and A L T , i.e. high degree o f overlap was observed between the elderly and the y o u n g groups. In order to confirm our initial observations, the sample sizes were increased. Analyses o f the larger sized sample groups o f y o u n g and elderly subjects again indicated that there was no statistical difference between the m o n o c y t e A H H activity in elderly and y o u n g subjects within experimental error using this experimental design. A L T activity was normal in both y o u n g and elderly population indicating no underlying liver dysfunction. The results also indicate that no significant statistical difference was observed for A L T between the y o u n g and elderly population within experimental error using this experimental design. As noted t h o u g h high intra g r o u p variability was observed for A L T in both the y o u n g and elderly population. A H H activity was~ however, clearly found to be statistically higher in smokers as c o m p a r e d to nonsmokers, supporting earlier conclusions drawn from drug model studies that drug
69 metabolizing enzymes are induced with smoking. In order to determine if smoking was affecting the monocyte A H H results in the aging population more so than in the young population the monocyte A H H activity was then statistically analyzed in elderly and young non-smoking populations. As indicated in the results section removal of this smoking population from both the young and elderly group lowered the mean monocyte AHH activity in both groups. The affect on the elderly population appeared to be greater; the mean monocyte AHH activity in the elderly group was reduced from 0.83 to 0.46 with removal of the non-smokers. Statistical analysis of the non-smoking young and elderly group again clearly indicated that there was no significant difference in monocyte AHH activity between the young and elderly non-smoking population within experimental error using this experimental design. Also removal of the non-smoking population from the total group significantly reduced the intra-group variability. In the elderly, several age related changes in anatomy and physiology have been found to correlate with alterations in hepatic metabolism of drugs in this population. These things include a reduction in liver size, a decrease in liver blood flow and an alteration in synthesis of drug binding proteins [3,12]. Wynne et al. [13] found a significant reduction in liver volume, apparent liver blood flow and perfusion and suggest this may at least partially account for the decline in the clearance of many drugs undergoing liver metabolism, particularly those of high extraction. The clearance of capacity limited drugs is influenced by the extent of plasma protein binding and, in general, those drugs (acidic) which bind to albumin tend to exhibit an increased free fraction, while those binding to a-acid glycoprotein tend to exhibit a decreased free fraction with age due to a decrease and increase, respectively, in these serum proteins [14]. This trend, however, has many exceptions and Vestal [15] suggests the clinical significance of age related changes is small and that other factors contribute in larger degree to inter-individual variability in protein binding. It has also been implied that age related changes occur due to an impairment in the activity of drug metabolizing enzymes. In the rat, particularly in the male, it has been well documented that the specific activities of a variety of hepatic microsomal monooxygenases fall with age both in relation to the content of microsomal protein and liver weight [16]. Recently Mote et al. [17] investigated the influence of age on liver gene expression and found a progressive decrease in basal Pi and P3-450 RNA levels between 4 and 28 months of age in BI0.RIII mice, but not C57.BL/10 mice. By using an antibody against the major 'male specific' species of cytochrome P-450, Fujita et al. [18] have provided evidence that differences in the amount of this isoenzyme (P-450ml) may be responsible for the age related and sex associated differences in some of the drug metabolizing activities in rats. He further proposes [18] that senescence associated feminization of the drug metabolizing ability of the male rat liver may be due in part to the decrease in testosterone levels in old age. Taken together with the work of Mote et al. [17] on age related changes in P-450 gene expression, an elegant animal model emerges for the senescence associated [9]change
70 of the relative abundance o f multiple species of P-450 in the liver into the female pattern. Caution is urged in the extrapolation of these changes to humans since the alterations in enzyme activity were species, strain, sex and substrate specific. This model, in addition, has not been corroborated by primate (Macacu monkey) models where no significant age related change was detected in the mixed function oxidase enzymes studied [19,201 nor has it been corroborated in humans [21,22]. In humans there is limited data on in vitro drug metabolizing enzyme capacity, while data from in vivo studies involving the mixed function oxidase system has been contradictory. In vivo studies using antipyrine as the model substrate have demonstrated an age associated reduction in the metabolic clearance for this drug [23]. Since antipyrine is a low extraction drug and is minimally bound to plasma proteins, its systemic clearance approximates drug metabolizing enzyme activity. It has hence been suggested that the age dependent decline in antipyrine clearance is indicative of a decline of the activities of the microsomal mixed function oxidase enzyme with age. Recent research [23], however, finds no evidence for a decrease in hepatic microsomal function with advancing age as measured by the aminopyrine breath test (n = 60). In addition, Van Gell and Van Bezooijen [24] found no changes with aging in the partial clearances of the antipyrine metabolites 3-hydroxymethylantipyrine (HMA) and 4-hydroxyantipyrine (OHA) in vivo, but a decrease from age 3 to 24 months followed by an increase up to age 36 months for norantipyrine (NORA) in rats. Thus, the collective data for age related changes in the mixed function oxidase system predicted by this model would seem ambiguous at best. In a study in which actual tissue samples were obtained at cholecystectomy or vagotomy from 17 patients, no correlation was observed between age and microsomal protein recovery, maximum enzyme activity or apparent enzyme activity [12]. Since this involved a select group of elderly patients fit enough for surgery, fears have been expressed about whether such findings can be generalized, especially with regard to the frail old. Our results for human male versus female A H H corroborate our earlier suggestion [6] that there is no sex related difference in human monocyte A H H activity nor is there any correlation between decreasing A H H activity and increasing age in these groups. As a result, our data for A H H activity as a whole suggests that the rodent models [17,18] of selective isozyme changes with aging may not be applicable to humans. In this study, MCM from both young and elderly subjects was shown to inhibit murine hepatocyte A H H , when compared to heat treated M C M samples, suggesting that release of a heat sensitive factor may be altering the A H H activity in certain individuals and may be contributing to population variability in A H H activity. One factor which may be acting to produce this effect is IL-1. 1L-1 has roles in immunity, inflammation and connective tissue metabolism and is made up of two subtypes, alpha and beta, with a common receptor on target cells [25]. In a study to determine the cause of endotoxin mediated depression of P-450
71 levels, Shedlofsky [26] showed significant depression of P-450 levels by injection of IL-1 into mice. Sujita [27] has found that cultured rat hepatocytes express a considerable amount of IL-1 receptor and that incubation of these hepatocytes with I L-1 for 18 h decreased the contents of P-450 in a dose dependent manner. Our results [28] have found that in the presence of antibody to IL-I, the inhibitory effect of MCM media on hepatocyte AHH activity is significantly reduced. It is interesting to note in this regard that Yamaguchi et al. [29] have found that the amount of IL-I released from LPS-stimulated alveolar macrophages was significantly decreased in smokers compared with that in non-smokers. Such a factor may be contributing to the markedly elevated monocyte AHH activity we have reported for smokers. Based on this evidence, then, there seems to be emerging a concept of a modulatory role of IL-I on drug metabolizing activity. In freshly isolated monocytes or macrophages derived from monocytes in vitro, Lewis et al. [30] have found a size dependent functional heterogeneity in the amount of IL-1 released per cell and in the number of cells secreting detectable levels of these products at one particular time. The different fraction of elderly subjects exhibiting a MCM inhibitory effect, as compared with young subjects, could indicate some sort of alteration in this heterogeneity with aging. Despite its lack of a direct triggering action, Cavaillon et al. [31] have found that human recombinant C5a is able to act synergistically with LPS, leading to higher TNF and IL-I release-by human monocytes. They propose that enhanced release of these two factors might occur during Gram-negative infections, since LPS is known to lead to C activation. Tatsuno et al. [ 32] found, moreover, a significant augmentation of IL-l-like activity in monocytes by leukotriene B4, while Laurenzii et al. [33] reported a similar effect for substance P. Taken together, these findings suggest there could be a number of intermediary factors modulating a possible alteration in the heterogeneity of IL-1 release with aging. As pointed out by Bradley et al. [34] it has long been postulated that deficiencies in the production of certain monokines could account for the diminished febrile responses and increased severity of infections in the elderly. They found, however, that while in vitro assays appeared to show a decrease in IL-I and TNF activity, endogenous pyrogen assays showed normal production of IL-I and TNF by macrophages of aged rats. One might speculate that it may be intermediary factors, like those referred to above, acting to trigger IL-I release in vivo, but not in vitro, which could be influencing blood monocytes like the macrophages in this study. Recently, Arend et al. [35] have further found that human monocytes cultured on adherent IgG produce a specific IL-I inhibitor that functions as a receptor antagonist (IL-ira) and suggest an important role for this substance in normal and pathophysiologicai states. This would seem to provide another alternative explanation for the decreased secretion of IL-I in aged macrophages found by Bradley et al. [34] in vitro and possibly reveals a further modulatory factor which may be deranged in aging. We have also found [28] that catalase, which inactivates activated oxygen species,
72
also reduces a component of the inhibitory effect of the MCM on hepatocyte AHH. Interestingly enough, a study by Ischiropoulos et al. [36] indicates that in rats, the proportion of the relatively inactive forms of Cu, Zn superoxide dismutase increase with age, while Niwa et al. [37] have shown that levels of lipid peroxides in elderly humans may be elevated as compared to the population in general (P < 0.05). Thus, altered activated oxygen disposition in aging may have an important role relating to drug metabolism. Azri and Renton [38] have speculated that the inhibition of P-450 involved in Listeria infections may be caused by the hemolysin produced by this organism acting to damage the mixed function oxidase system. Renton and Knickle [39] have shown that interferon inducers depress the m R N A for the P-450LA isoenzyme and suggest a role for interferon mediated P-450 depression in viral infections. Hence, a complex network of factors, whose specific mode of interaction remains to be worked out, may be operating to produce the inhibitory effect observed in our studies. The fact that the elderly population had a larger fraction of subjects with an MCM inhibitory effect suggests that the interactions in this group may differ as compared with young controls, at least as pertains to the subjects in this study. In conclusion we have shown: (1) mean monocyte A H H activity does not show a statistically significant change with aging; (2) A H H activity is statistically significantly higher for smokers; (3) no statistical difference exists in monocyte A H H activity between young or old males and females; (4) the existence of an inhibitory effect of MCM media on hepatocyte AHH, present in similar degree, but with a greater prevalence, in the elderly as compared to the control population. A C K N O W L E D G E M ENTS
Thanks to Richard A. Isbrucker for his invaluable technical assistance, Dr. David Hogan for his aid in securing the geriatric population, the Dalhousie Medical Research Foundation for studentship support (A.D.P.) and Lorna Russell for secretarial assistance. This study was supported by the Medical Research Council of Canada and the Dalhousie Medical Research Foundation. REFERENCES 1 2 3
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