Plasma concentration of myeloperoxidase enzyme in pre- and post-climacterial people: related superoxide anion generation

Experimental Gerontology 37 (2001) 137±148

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Plasma concentration of myeloperoxidase enzyme in pre- and postclimacterial people: related superoxide anion generation GaÂbor BeÂkeÂsi a,*, ReÂka Kakucs a, JoÂzsef SaÂndor b, EnikoÍ SaÂrvaÂry c, Ibolya Kocsis a, Detlef Sprintz a, Szabolcs VaÂrbõÂro d, ZoltaÂn Magyar e, AndraÂs HrabaÂk f, JaÂnos FeheÂr a, BeÂla SzeÂkaÂcs a a

2nd Department of Medicine, Semmelweis University, SzentkiraÂlyi Utca 46, 1088 Budapest, Hungary Department of Blood Bank of PeÂterfy Hospital, PeÂterfy S. Utca 8±20, 1076 Semmelweis University, Budapest, Hungary c Department of Transplantation, Semmelweis University, Baross G. Utca 23±25, 1082 Budapest, Hungary d È lloÈi uÂt 78/A, 1082 Budapest, Hungary 2nd Department of Gynecology, Semmelweis University, U e 1st Department of Obstetrics, Semmelweis University, Baross G. Utca 27, 1088 Budapest, Hungary f Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Puskin Utca G, 1088 Budapest, Hungary b

Received 19 December 2000; received in revised form 12 June 2001; accepted 11 July 2001

Abstract Neutrophil granulocytes are involved in the pathogenesis of atherosclerosis also through their free radical generation. The aim of the study was to test how extracellular levels of myeloperoxidase (MPO; a granulocyte enzyme playing role in free radical production) change by age and what effect this change has on the production of the free radical superoxide anion by neutrophils. We also wanted to examine whether the antioxidant effect of different steroid hormones is realized through the MPO. Plasma myeloperoxidase concentrations of healthy blood donors were quanti®ed by ELISA. Superoxide anion production was measured by photometry. Myeloperoxidase concentration was signi®cantly lower in plasmas obtained from older women and men than in those from younger subjects. Adding the MPO inhibitors 4-aminobenzoic acid hydrazide (ABAH) and indomethacin to the granulocytes, the generation of superoxide anion increased and the decreasing effect of the steroids on superoxide production was inhibited. Incubating the neutrophils with the product of the reaction catalyzed by MPO itself (hypochlorite anion), we found signi®cant decrease in superoxide generation. According to our results MPO seems to diminish the production of superoxide anion and so probably has an antioxidant ability. Therefore, its lower plasma levels may contribute to the increasing incidence of atherosclerosis and other free radical mediated disorders in old people. Thus, after further studies MPO might become one of the indicators of cardiovascular risk and the scavenger capacity in general. q 2001 Elsevier Science Inc. All rights reserved. Keywords: Neutrophil granulocytes; Myeloperoxidase enzyme; Myeloperoxidase inhibitors; Superoxide anion; Atherosclerosis; Steroids; Antioxidant effect; Vasoprotection

1. Introduction * Corresponding author. Tel.: 136-1-317-4548; fax: 136-1-2660816. E-mail address: [email protected] (G. BeÂkeÂsi).

Experimental and clinical studies show that the corpuscular elements of blood, in particular white blood cells, play an important role in the pathomechanism of

0531-5565/01/$ - see front matter q 2001 Elsevier Science Inc. All rights reserved. PII: S 0531-556 5(01)00151-6

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Fig. 1. Free radical reactions in neutrophil granulocytes. Reactive oxygen species are in dark squares (downwards: superoxide anion, hydrogen peroxide, hypochlorite anion, hydroxyl radical, singlet oxygen, nitric oxide, peroxynitrite, nitronium ion and nitrogen dioxide). The light squares indicate enzymes (downwards: NADPHoxidase, superoxide dismutase and myeloperoxidase). The thick arrows show catalyzing effects and the thin ones the directions of the reactions. The ®gure demonstrates that superoxide anion is source of many other reactive oxygen intermediates. In the fourth reaction instead of molecular oxygen singlet oxygen can also be produced (under each other). In the last reaction the divergent arrows indicate the possibilities of decomposition of peroxynitrite. Our ®gure also shows the increasing effect of steroids on MPO and the inhibiting in¯uence of MPO-hypohalite system on superoxide anion generating NADPH-oxidase.

atherosclerosis (Elneihoum et al., 1997). Moreover, the elevated production of free radicals is a major factor in the abnormal interaction of leukocytes and endothelial cells (Das, 1992; MohaÂcsi et al., 1996). One of the most relevant markers of neutrophil activity is the release of MPO which takes place in several free radical reactions (King et al., 1997; Podrez et al., 2000). It is an abundant heme protein secreted from activated phagocytes. MPO catalyzes

the oxidation of halide ions to hypohalous acids at the expense of hydrogen peroxide.Their normal functions include host defense and surveillance. However, under certain circumstances an excess of these oxidizing species can overwhelm local antioxidant defenses and lead to oxidant stress and oxidative tissue injury, processes implicated in the pathogenesis of some diseases as for example atherosclerosis. MPO is one pathway for protein and lipoprotein oxidation, however, the biological consequences of the MPOcatalyzed reactions in vivo are still unclear. MPO contains as its prosthetic group the same heme that is found in hemoglobin (Babior, 2000). Unlike hemoglobin, however, MPO is green, not red, and is the substance that imparts the greenish color to pus. MPO de®ciency is an inherited condition, affecting 1 of 500±2000 people. It can also be rarely symptomatic. In those subjects in whom MPO de®ciency is clinically manifest, it appears as a heightened susceptibility to candida infections, particularly in patients who have MPO de®ciency and diabetes mellitus. In most of the previous publications the higher level of MPO was connected to enhanced free radical production and greater exposure to atherosclerosis (MohaÂcsi et al., 1996; Dougherty et al., 1994). However, other authors have described that increased MPO activity also contributes to the termination of the free radical production by neutrophils through inactivation of NADPH-oxidase enzyme generating superoxide anion (Jandl et al., 1978; cited by Feher et al. (1987). Although the free radical superoxide anion is reactive only to a limited degree, it also induces peroxidatic changes in lecithin-cholesterol membranes (Seligman et al., 1979) and can be transformed into other more reactive oxygen intermediates, so it is of great importance (Fig. 1). By cooperating with hydrogen peroxide, superoxide anion can produce hydroxyl radical and singlet oxygen, which are the proximate causes of lipid peroxidation (Kellogg and Fridovich, 1977). Beyond this role of superoxide anion it is a vasoconstrictor (Das, 1992) and signi®cantly stimulates vascular smooth muscle cell proliferation in aortic explant of rats (Cathapermal et al., 1998). By neutralizing the vasodilator nitric oxide (NO) superoxide anion produces peroxynitrite, which also oxidizes the lipoproteins, decomposes to highly reactive species (Van Der Vliet et al.,

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1994) and contributes to the loss of cardiac ef®ciency in animal heart (Ferdinandy et al., 1999). Females of reproductive age with normal menstruation and sex hormone production enjoy relative 'immunity' from atherosclerosis (Selzeman et al., 1998). After the onset of the menopause, however, this protection gradually decreases or vanishes, and the risk of progressive vascular damage increases (Matthews et al., 1989). According to our previous data post-menopausal women and older men have lower intracellular MPO activity than the younger subjects (BeÂkeÂsi et al., 2001). Furthermore, using estrogen replacement therapy, the intracellular MPO activity and spontaneous release of MPO from isolated neutrophil granulocytes increased (BeÂkeÂsi et al., 2001). According to another publication human PMN-s showed increased MPO release after hydrocortison administration (Hetherington and Quie, 1985). On the other hand, there has not been any information concerning the similar effect of progesterone, testosterone and aldosterone in literature as regards to this. Moreover, we managed to prove that the in vitro adding of MPO to granulocyte suspensions signi®cantly reduced their superoxide anion production (BeÂkeÂsi et al., 2001). Besides, except aldosterone all end-steroids decrease the superoxide anion generation by neutrophil granulocytes (BeÂkeÂsi et al., 2000). 4-Aminobenzoic acid hydrazide (ABAH) is a speci®c MPO inhibitor (Mytar et al., 1999). MPO oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous MPO reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation (Kettle et al., 1997). Indomethacin has also been known as a strong MPO inhibitor. It inhibits directly the chlorinating activity of the enzyme (Shacter et al., 1991). The increasing incidence of atherosclerosis and other free radical mediated disorders (tumors, pulmonary emphysema, the process of aging itself, etc.) in older people has been widely pointed out (Feher et al., 1987). According to a relevant article (Chan et al., 1998) the activity of polymorphonuclear leukocytes (PMN) from human subjects over 60 years was signi®cantly lower than the activity measured in younger subjects. At the same time the PMN chemiluminescence (an instrument of estimating free radical production by leukocytes) produced by

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stimulation was found to be positively correlated with the increasing age of human subjects. The aim of our study was to test how plasma levels of myeloperoxidase change by age. Furthermore, we wanted to clarify what in¯uence this change had on superoxide production by neutrophils and whether the antioxidant effect of steroid hormones is realized through MPO. 2. Materials and methods Plasma myeloperoxidase concentration was measured in the blood samples of 106 healthy blood donors. These were left over samples of blood obtained in order to determine the clinical parameters needed for giving blood. Comparisons between age groups and sexes were made only on the basis of data from non-smoking male and female subjects (aged 20±40 and 50±70 years old), who were not treated with any medicine. Blood was drawn from a forearm vein using EDTA vacutainer tubes. The blood was centrifuged at 2000 rpm for 20 min. An aliquot of the plasma was frozen at 2208C. An ELISA method was used to quantify plasma MPO levels (MPO-kit, Bioxytech-Oxis International Inc., Portland, OR, USA). One hundred ml of the tenfold diluted plasma from different subjects was incubated in the wells of a sectionable microplate. The results were given in ng/ ml. In order to measure the superoxide anion production we had to obtain blood samples of healthy volunteers into EDTA-tubes. The blood was applied to Histopaque (Sigma, 1077-1) in layers for the sedimentation of red blood cells and put aside for an hour. Then it was transferred to 63 and 72% Percoll (Pharmacia, 170891-01) and centrifuged for 25 min at a rate of 300 £ g at 208C. Granulocytes thus separated were buffer-washed twice and centrifuged at 220 £ g: Cells aggregated at the bottom of the tube were resuspended in a few milliliters of buffer (Hank's salt solution, Biochrom KG Berlin, cat. numb.:2035), then counted using Turk's solution. The cell concentration of the suspension was adjusted to 5 million/milliliter. The granulocyte-suspensions were incubated for 2 h at 378C alone with two concentrations (50±100 mmol) of MPO inhibitor ABAH (Sigma, A-41909), also with two concentrations (15±40 mmol) of the other MPO

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Fig. 2. Plasma concentration of MPO in samples of females and males aged 20±40 vs. 50±70. Values are means ^ SEM (standard error of the mean) (see in columns).

inhibitor indomethacin (Sigma 17378), with three concentrations (1±10±100 mmol) of sodium hypochlorite (NaClO) (hypochlorite anion is the product of the reaction catalyzed by MPO) (Sigma 23,930-5), separately with 10 27 M concentration of steroid hormones (17-b-estradiol, Sigma E8875; progesterone, Sigma P0130; testosterone, Fluka 86500; hydrocortisone, Sigma H4001; d-aldosterone, Sigma A6628; cortexolone, Sigma R0500; dehydroepiandrosterone-DHEA-, Sigma D4000; and dehydroepiandrosterone sulfate-DHEAS-, Sigma D5297. The last two hormones were used in a 10 25 M concentration.) and with hormones and ABAH or indomethacin together. During our previous work mentioned above (BeÂkeÂsi et al., 2000) we used three concentrations of the steroids (1027 ±1028 and 1029 M). In this trial we did not want to repeat the whole procedure, we only used the highest concentration. Otherwise, because of the MPO inhibitor samples we should have obtained much more blood from our volunteers. This would have been too much of a burden for them. When determining the spontaneous MPO release from testosterone treated volunteers' granulocytes we also used an ELISA method after the above mentioned isolation of the cells. Polyclonal antiMPO antibody (Dako A 398) was applied as a 'coat', whereas MPO-standard was bought from Calbiochem. The substrate mixture contained TMB (tetramethylbenzidine, Sigma), DMSO (dimethyl

sulfoxide, Sigma), substrate buffer (Sigma P-4922) and 1% H2O2. The results were expressed in mU/ml. The superoxide anion production of neutrophil granulocytes was measured using the method of Quarneri et al. (1990) as modi®ed by Jansson (1990). The quantity of free radicals released was assessed by photometry; the reduction of ferricytochrome-C (Sigma C-752) by the superoxide was measured as optical density (OD). The maximum superoxide anion production was assessed as change of optical density (DOD) 5 min after being stimulated with FMLP (N-Formyl-Met-Leu-Phe, Sigma F 3506). After 5 min there were no cases of further elevations of OD values. The result of the ®rst measurement just after adding FMLP to the cell suspension served as the starting value (zero point). Further measurements were carried out every minute (0±1±2±3±4±5 min). Intracellular MPO activity (mean peroxidase index, MPXI) was measured using a Technicon H-3 automat (distributor: Bayer AG). In brief, adding hydrogen peroxide and chromogen to neutrophil granulocytes yields a dark precipitate when peroxidase activity is present. Peroxidase activity is proportional to the intensity of staining, which is measured as the absorption of light by MPO-containing cells passing through a channel illuminated by a wolfram bulb. The machine automatically measures MPO activity in addition to WBC and CBC. The mean peroxidase index is a mathematical description of the distance of the actual centre of the observed neutrophil cluster from the theoretical center

G. BeÂkeÂsi et al. / Experimental Gerontology 37 (2001) 137±148 Table 1 Superoxide generation (DOD) of human neutrophil granulocytes incubated with different steroids and different concentrations (50± 100 mmol) of ABAH

Control ABAH 50 ABAH 100 Cortisol Cortisol 1 ABAH 50 Cortisol 1 ABAH 100 Aldosterone Aldosterone 1 ABAH 50 Aldosterone 1 ABAH 100 Cortexolone Cortexolone 1 ABAH 50 Cortexolone 1 ABAH 100 17-b-Estradiol 17-b-Estradiol 1 ABAH 50 17-b-Estradiol 1 ABAH 100 Progesterone Progesterone 1 ABAH 50 Progesterone 1 ABAH 100 Testosterone Testosterone 1 ABAH 50 Testosterone 1 ABAH 100

Mean

SD

0.178 0.206 0.215 0.159 0.206 0.199 0.190 0.210 0.217 0.190 0.215 0.214 0.152 0.214 0.199 0.155 0.194 0.207 0.145 0.207 0.207

0.107 0.110 0.101 0.100 0.099 0.094 0.116 0.099 0.099 0.110 0.097 0.104 0.104 0.109 0.100 0.091 0.124 0.101 0.094 0.094 0.101

of an idealized neutrophil cluster data set (normal range: between 210 and 110). Statistical analysis of ABAH-indomethacin-and sodium hypochlorite-data was performed using repeated measurement ANOVA-method with setting of contrast values to the control (SAS v. 6.12 statistical program package). LSD-method was used as a post hoc test. The two-way ANOVA method was applied for comparison of the superoxide anion generation by neutrophil cells of young and old donors. During the processing of plasma MPO values and data received after testosteron and prednisolon treatment we used two and one sample Student's test. Signi®cance level was set to p , 0:05: Results were given as means ^ SD (standard deviation). The error bars represent errors between individuals. The trial protocol was approved by the local research ethics committee. 3. Results Myeloperoxidase concentration was signi®cantly

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lower in plasmas obtained from women aged 50±70 years old …n ˆ 20† than in those from females aged 20±40 years old …n ˆ 41† (28:11 ^ 10:25 vs. 44:30 ^ 21:29 ng=ml; p ˆ 0:0021),(see Fig. 2). Similar difference was observed when two male groups of matching age were compared (28:68 ^ 10:62 ng=ml; n ˆ 20 vs. 42:01 ^ 16:13 ng=ml; n ˆ 25; p ˆ 0:0027), (see Fig. 2). No signi®cant difference was found between gender groups of matching age (p ˆ 0:8657; p ˆ 0:6463). Incubating the isolated neutrophil cells with two concentrations of ABAH, we observed signi®cant increases of the superoxide anion production (p , 0:001; n ˆ 17; females and males aged 22±72 years old). (This was found not only during incubation of the cells with ABAH alone but also together with all steroids.) In accordance with our previous, abovementioned data (BeÂkeÂsi et al., 2000) incubation of the granulocytes with 10 27 M concentrations of cortisol, 17-b-estradiol, progesterone and testosterone yielded signi®cant reductions of superoxide generation in comparison with the control samples (in the case of the sex steroids: p , 0:001; cortisol: p ˆ 0:0049). Aldosterone and cortexolone did not have such an effect this time either (p ˆ 0:43; p ˆ 0:46). Combining ABAH with single steroids during the incubation process, the antioxidant effect of the four hormones proved to be signi®cantly reversible, furthermore, adding ABAH and aldosterone or cortexolone to the cells also showed signi®cant increase in superoxide production (in the case of combined incubation of ABAH with cortisol, estrogen, progesterone and testosterone: p ˆ 0:0001; with aldosterone and cortexolone: p , 0:05). There was no signi®cant difference between samples containing 50 and 100 mM ABAH regarding the superoxide generation (p . 0:05 in all cases). Comparing the superoxide levels of samples originating from young and old donors, we did not ®nd signi®cant differences …p ˆ 0:5738†; but there was interestingly an increasing tendency in samples of elderly subjects at all points (presumably because of their lower sex hormone and MPO levels). Data are listed in the Table 1 (see also Figs. 3(A) and (B)). Incubating the neutrophils of seven healthy volunteers (females and males aged 35±52 years old) with two concentrations of the other MPO inhibitor indomethacin, we observed the same phenomenon. When

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Fig. 3. (A) Superoxide anion production of human neutrophil granulocytes incubated with two concentrations (50±100 mmol) of MPOinhibitor ABAH, separately with 10 27 M concentration of different steroid hormones and a combination of both ABAH and steroids. (B) Superoxide anion production of human neutrophil granulocytes incubated with two concentrations (50±100 mmol) of MPO inhibitor ABAH, separately with 10 27 M concentration of different steroid hormones and a combination of both ABAH and steroids. The ®gure also shows the not signi®cantly higher mean superoxide anion generation of older individuals at all points. (young: 22±45 years; elderly: 45±72 years).

we added indomethacin on its own as well as together with steroids to the cells it resulted in a signi®cant increase of superoxide release (control vs. indomethacin15: p ˆ 0:0012; control vs. indomethacin40: p ˆ

0:0001; in the case of combined incubation with cortisol, estrogen, progesterone and testosterone: p , 0:001; with DHEA and DHEAS: p , 0:05). After the incubation of the cells with 10 25 M concentration

G. BeÂkeÂsi et al. / Experimental Gerontology 37 (2001) 137±148 Table 2 Superoxide generation (DOD) of human neutrophil granulocytes incubated with different steriods and different concentrations (15± 40 mmol) of indomethacin

Control Indomethacin 15 Indomethacin 40 Cortisol Cortisol 1 indomethacin 15 Cortisol 1 indomethacin 40 Estrogen Estrogen 1 indomethacin 15 Estrogen 1 indomethacin 40 Progesterone Progesterone 1 indomethacin 15 Progesterone 1 indomethacin 40 Testosterone Testosterone 1 indomethacin 15 Testosterone 1 indomethacin 40 DHE DHE 1 indomethacin 15 DHE 1 indomethacin 40 DHES DHES 1 indomethacin 15 DHES 1 indomethacin 40

Mean

SD

0.116 0.131 0.135 0.092 0.119 0.125 0.102 0.123 0.127 0.107 0.131 0.141 0.093 0.117 0.130 0.090 0.121 0.127 0.066 0.085 0.109

0.061 0.064 0.062 0.049 0.061 0.064 0.058 0.063 0.059 0.060 0.063 0.077 0.062 0.064 0.071 0.053 0.050 0.070 0.061 0.051 0.078

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Table 3 Superoxide generation (DOD) of human neutrophil granulocytes incubated with different concentrations (1±10±100 mmol) of NaClO

Control NaClO 1 NaClO 10 NaClO 100

Mean

SD

0.109 0.109 0.076 0.062

0.033 0.032 0.024 0.025

of DHEA and DHEAS on their own we also found a signi®cant reduction in superoxide production (p ˆ 0:0008; p ˆ 0:0011). We did not ®nd any dose dependency in this part of our investigations either (p . 0:05 in all cases). (see Table 2 and Fig. 4). The incubation of the neutrophil cells of ®ve healthy volunteers (females and males aged 22±52 years old) with two concentrations of NaClO yielded signi®cant decrease of superoxide release (control vs. NaClO1: p ˆ 0:9632; control vs. NaClO10: p ˆ 0:0001; control vs. NaClO100: p , 0:0001). In contrast to the MPO inhibitor investigations on this occasion we found signi®cant dose dependency …p # 0:001†: Not more than 1±2% of the cells died during

Fig. 4. Superoxide anion production of human neutrophil granulocytes incubated with two concentrations (15±40 mmol) of MPO inhibitor indomethacin alone and separately with 10 27 and 10 25 M (DHEA and DHEAS) concentrations of different steroid hormones.

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Fig. 5. Superoxide anion production of human neutrophil granulocytes incubated with three concentrations (1±10±100 mmol) of NaClO.

the isolation and incubation process in the control as well as the treated samples. (see Table 3 and Fig. 5). In order to clarify whether not only estradiol but also testosterone has an increasing effect on the MPO release from the neutrophils, we carried out a supplementing investigation. Seven male volunteers (all of

them are doctors and co-authors of this article) took 40 mg testosterone undecanoicum three times a day (Andriol, Orgnon) for 12 days. (During the investigation of post-menopausal women Ð in the above mentioned publication (BeÂkeÂsi et al., 2001) Ð the blood samples of individuals were also evaluated

Fig. 6. Spontaneous release of MPO from human neutrophil granulocytes before and after 12 day testosterone administration. Values are means ^ SEM:

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Fig. 7. Intracellular MPO activity (MPXI) of human neutrophil granulocytes before the injection of 25 mg prednisolone and 2 h later. Values are means ^ SEM:

after 12 day estrogen replacement therapy.) The spontaneous release of MPO from isolated granulocytes was signi®cantly higher after testosterone administration in comparison with the control values (439:11 ^ 145:22 vs. 602:81 ^ 166:28 mU=ml; p , 0:005).(see Fig. 6). We have also completed the available knowledge concerning the cortisol-MPO relation by an additional measurement. Four of our healthy co-authors (men and women) were voluntarily injected intravenously by 25 mg prednisolone sodium succinate (Di-Adreson F aquosum, Organon). The MPXI values determined two hours after the injection proved to be signi®cantly higher than the control ones (0:8 ^ 0:97 vs. 2:68 ^ 1:18; p , 0:05). (see Fig. 7). 4. Discussion There are several publications supporting the opinion that MPO is involved in the atherogenesis through its ability to produce free radicals. MPO is reported to be present in human atherosclerotic lesions (Dougherthy et al., 1994). MPO release from neutrophils was found to be signi®cantly higher after fMLP stimulation in atherosclerotic patients and healthy old subjects than in healthy middle-aged

people. At the same time the resting level of superoxide anion was increased in atherosclerotic patients and healthy old individuals (MohaÂcsi et al., 1996). Myeloperoxidase mediated oxidation of LDL has been considered an important mechanism by which LDL undergoes oxidation in vivo (Wieland et al., 1993; Savenkova et al., 1994). Our ®ndings are not consistent with these results, in addition, there have been no studies dealing with changes of plasma concentration of MPO by age up to now in literature. Our results show that MPO plasma concentrations are signi®cantly lower in healthy old females and males than in young groups. Furthermore, in our previous in vitro experiment a signi®cant reduction of superoxide anion level was found when adding MPO to neutrophil granulocyte suspensions isolated from 20 normal female and male volunteers aged 30± 65 years old (BeÂkeÂsi et al., 2001). On the same occasion we could verify that older individuals have lower intracellular MPO activity (MPXI) than the younger ones. Besides, we have described that during estrogen replacement therapy both the spontaneous MPO release and the MPXI increase. According to our other article (BeÂkeÂsi et al., 2000) estrogen, progesterone, testosterone and cortisol are able to reduce the superoxide generation by neutrophils. It is logical to

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ask whether the scavenger ability of these steroids is displayed through MPO. In this paper we demonstrated that not only estradiol but according to our preliminary data also testosterone and cortisol have the capacity to enhance MPO release and activity. Moreover, we proved in this article that MPO inhibitors ABAH and indomethacin increased the superoxide generation and reversed the antioxidant effect of the steroid hormones. Our DHEA and DHEAS data (these steroids also decreased superoxide release from neutrophil cells) are in line with the results of other authors (Rom and Harkin, 1991) who reported similar ®ndings using 10 25 M concentrations of the hormones and alveolar macrophags. Furthermore, we also incubated the neutrophils of healthy volunteers with NaClO (CLO 2 is the product of the reaction catalyzed by MPO) and after two hours we found a signi®cant reduction of superoxide anion generation. Since myeloperoxidase seems to inhibit the generation of superoxide anion, the lower plasma levels of MPO found in older subjects might contribute to the development of some free radical mediated disorders such as onco- and atherogenesis affecting mostly the elderly population. This conclusion may also be supported by other authors (Jandl et al., 1978). They assumed that enhanced MPO activity was also involved in the termination of the generation of free radicals by neutrophils. Accordingly, the MPO-H2O2-halide system may inactivate the NADPH-oxidase system. (They did not draw further clinical-practical conclusions from this ®nding.) Our results presented evidence that proved the point directly. According to our new data not the 'peroxide-halide-myeloperoxidase mechanism' in general but precisely the product of the third recation in a chain of reactions, the product of MPO has the superoxide inhibiting activity by a negative feed-back effect. This is a really new theoretical aspect of the regulatory mechanism of free radical production in leukocytes. During our investigations we reconstructed the physiologically lower MPO plasma concentration of elderly individuals. NADPH-oxidase is the ®rst enzyme of the pathway (see Fig. 1) the third reaction of which is catalyzed by MPO itself. Evidently, the inhibition of the ®rst step in a chain of reactions has a rate-limiting effect on the whole process, or in this

case, it decreases the production of superoxide anions as well as the other reactive oxygen species (e.g. H2O2, CLO 2, etc.) playing role in the pathomechanism of several diseases. In the group of patients with MPO de®ciency (Badwey and Karnovsky, 1980; Klebanoff, 1980) activation of the `respiratory burst', increased oxygen consumption and augmented production of superoxide anions, H2O2 and toxic oxygen species are observable. Enhanced MPO activity is associated with attenuated superoxide anion production in the early phase of acute pancreatitis (Ferencz et al., 1997). MPO itself also combines with superoxide anion and forms an inactive compound (King et al., 1997).The positive consequence of this is obvious. The increased level of MPO may further be bene®cial, because myeloperoxidase-modi®ed HDL has been reported to increase in vitro cholesterol ef¯ux from macrophage-derived foam cells (Zuckerman and Bryan, 1996). Although myeloperoxidase has been demonstrated to oxidize LDL (Wieland et al., 1993; Savenkova et al., 1994; Santanam and Parthasarathy, 1995), on the other hand, MPO might have an important antioxidant effect after all through decreasing the superoxide anion level. Our new conceptual model also gives an explanation for the enhanced risk of infections during glucocorticoid administration (the superoxide anion is a source of bactericidal compounds) and for the advantageous effect of hormonal replacement therapy in case of postmenopausal hypertension ( the superoxide anion is a potent vasoconstrictor) (Szekacs et al., 2000). Summarizing other and our previous and present data steroids seem to be able to enhance the MPO level and activity probably through the in¯uence on MPO gene (Yamada et al., 1993). These authors identi®ed estrogen regulated DNA sequences involved in the expression of the MPO gene. Hereby (through the superoxide inhibiting property of MPO which could be very advantageous from the point of view of the vasoprotection and other free radical mediated disorders) they appear as potent antioxidant agents. The decreasing levels of sex steroids from the age of about ®fty might be reasons for diminished MPO and therefore increased superoxide levels, which

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may be responsible for the increasing development of atherosclerosis in the elderly age groups. One could perhaps suppose that sex steroids also have the feature of preserving the vascular youthfulness of man. These data may provide further arguments supporting the inevitably many-sided advantages of hormonal replacement therapy not only in women but also in both genders (Matthews et al., 1989; BeÂkeÂsi et al., 2000; Collins, 1999 Ð low plasma levels of testosterone in males are associated with increased coronary heart disease). According to the aforementioned ®ndings MPO may have an antioxidant function and it seems to be one of the indicators of age and gender dependent cardiovascular risk. Thus, pharmacological modulation of MPO activity might also provide new perspectives in the prevention of atherosclerosis and other free radical mediated diseases, and after further studies measurement of MPO levels may become a new laboratory approach to the forecast of the cardiovascular threat. Acknowledgements The authors wish to express their gratitude to Prof. Dr Zsolt Tulassay and Prof. Dr AndraÂs Falus for their expert advice, to Mrs Krisztina Nagy, Mrs MaÂrta Lakatos, Mrs EÂva ZaÂtonyi, Mrs Brigitte Mai and Mrs Zsuzsanna BaÂcsi for their devoted efforts in solving technical problems in laboratory work. The study was sponsored by the Maecenator Foundation and by Grant ETT 80-4/1998. References Babior, B.M., 2000. Phagocytes and oxidative stress. Am. J. Med. 109/1, 33±44. Badwey, J., Karnovsky, M., 1980. Active oxygen species and the functions of phagocytic leukocytes. Annu. Rev. Biochem. 49, 695±726. BeÂkeÂsi, G., Kakucs, R., VaÂrbõÂroÂ, Sz., RaÂcz, K., Sprintz, D., FeheÂr, J., SzeÂkaÂcs, B., 2000. In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes. Steroids 65/12, 889±894. BeÂkeÂsi, G., Kakucs, R., VaÂrbõÂroÂ, Sz., FeheÂr, J., Pazmany, T., Magyar, Z., Sprintz, D., SzeÂkaÂcs, B., 2001. Induced myeloperoxidase activity and related superoxide inhibition during hormone replacement therapy. Br. J. Obstet. Gynaec. 108, 474±481.

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Cathapermal, S., Lavigne, M.C., Leong-Son, M., Alibadi, T., Ramwell, P.W., 1998. Stereoisomerspeci®c inhibition of superoxide anion-induced rat aortic smooth muscle cell proliferation by 17 beta-estradiol is estrogen receptor dependent. J. Cardiovasc. Pharmacol. 31 (4), 499±505. Chan, S.S., Monteiro, H.P., Deucher, G.P., Abud, R.L., Abuchalla, D., Junqueira, V.B., 1998. Functional activity of blood polymorphonuclear leukocytes as an oxidative stress biomarker in human subjects. Free Rad. Biol. Med. 24 (9), 1411±1418. Collins, P., 1999. Vascular effects of ovarian hormones. Climacteric 2 (Suppl. 1), 47. Das, U.N., 1992. Can free radicals induce coronary vasospasm and acute myocardial infarction? Med-Hypotheses 39 (1), 90±94. Dougherthy, A., Dunn, J.L., Ratery, D.L., Heinecke, J.W., 1994. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J. Clin. Invest. 94, 437±444. Elneihoum, A.M., Falke, P., Hedblad, B., Lindgrade, F., Ohlsson, K., 1997. Leukocyte activation in atherosclerosis, correlation with risk factors. Atherosclerosis 131 (1), 79±84. Feher, J., Csomos, G., Vereckei, A., 1987. Free Radical Reactions in Medicine. Springer, Berlin. Ferdinandy, P., Panas, D., Schulz, R., 1999. Peroxynitrite contributes to spontaneous loss of cardiac ef®ciency in isolated working rat hearts. Am. J. Physiol. 276:H, 1861±1867. Ferencz, A., RoÍth, E., GaÂl, I., 1997. The role of neutrophil activation and in¯ammatory mediators in the pathomechanism of acute pancreatitis. Fifth Free Radical Research Conference, GoÈdoÈlloÍ, Hungary, 62. Hetherington, S.V., Quie, P.G., 1985. Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: function and granule protein content. Am. J. Hem. 20 (3), 235±246. Jandl, R.C., AndreÂ-Schwartz, J., Borges-Dubois, L., Kipness, R.S., Mc Murrich, B.J., Babior, B.M., 1978. Termination of the respiratory burst in human neutrophils. J. Clin. Invest. 61, 1176±1185. Jansson, G., 1990. Oestrogen induced enhancement of myeloperoxidase activity in human polymorphonuclear leukocytes Ð a possible cause of oxidative stress in in¯ammatory cells. Free Rad. Res. Com. 14, 195±208. Kellogg, E.W., Fridovich, I., 1977. Liposome oxidation and erythrocyte lysis by enzimically generated superoxide and hydrogen peroxide. J. Biol. Chem. 252 (19), 6721±6728. Kettle, A.J., Gedye, C.A., Winterbourn, C.C., 1997. Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide. Biochem. J. 321, 503±508. King, C.C., Jefferson, M.M., Thomas, E.L., 1997. Secretion and inactivation of myeloperoxidase by isolated neutrophils. J. Leukoc. Biol. 61, 293±302. Klebanoff, S., 1980. Oxygen metabolism and the toxic properties of phagocytes. Ann. Intern. Med. 93, 480±489. Matthews, K.A., Meilahn, E., Kuler, L.H., Kelsey, S.F., Caggiula, A.W., Wing, R.R., 1989. Menopause and risk factors for coronary heart disease. N. Engl. J. Med. 321, 641±646. MohaÂcsi, A., Kozlowszky, B., Kiss, I., Seres, I., FuÈloÈp, T., 1996. Neutrophils obtained from obliterative atherosclerotic patients

148

G. BeÂkeÂsi et al. / Experimental Gerontology 37 (2001) 137±148

exhibit enhanced resting respiratory burst and increased degranulation in response to various stimuli. Biochim. Biophys. Acta 1316 (3), 210±216. Mytar, B., Siedlar, M., Woloszyn, M., Ruggiero, I., Pryjma, J., Zembala, M., 1999. Induction of reactive oxygen intermediates in human monocytes by tumor cells and their role in spontaneous monocyte cytotoxicity. Br. J. Cancer 79 (5/6), 737±743. Podrez, E.A., Abu-Sound, H.M., Hazen, S.L., 2000. Myeloperoxidase-generated oxidants and atherosclerosis. Free Rad. Biol. Med. 28 (12), 1717±1725. Quarneri, C., Melandri, G., Caldarera, I., 1990. Reduced oxidative activity of circulating neutrophils in patients after myocardial infarction. Cell Biochem. Funct. 8, 157±162. Rom, W.N., Harkin, T., 1991. Dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis. Environ. Res. 55, 145±156. Santanam, N., Parthasarathy, S., 1995. Paradoxical actions of antioxidants in the oxidation of low density lipoprotein by peroxidases. J. Clin. Invest. 95, 2594±2600. Savenkova, M.L., Mueller, D.M., Heinecke, J.W., 1994. Tyrosyl radical, generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein. J. Biol. Chem. 269, 20,394±20,400. Seligman, M.L., Mitamura, J., Shera, N., Demopoulos, H.B., 1979. Corticosteroid (methylprednisolon) modulation of photoperoxidation by ultraviolet light in liposomes. Photochem. Photobiol. 29, 549±558.

Selzeman, C.H., Whitehil, T.A., Shames, B.D., Pulido, E.S., Cain, B.C., Harken, A.H., 1998. The biology of estrogen-mediated repair of cardiovascular injury. Ann. Throracic Surg. 65, 868± 874. Shacter, E., Lopez, R.L., Pati, S., 1991. Inhibition of the myeloperoxidase-H2O2-Cl- system of neutrophils by indomethacin and other non-steroidal anti-in¯ammatory drugs. Biochem. Pharmacol. 41, 975±984. Szekacs, B., Vajo, Z., Acs, N., Hada, P., Csuzi, L., Bezeredi, J., 2000. Hormone replacement therapy reduces mean 24-hour blood pressure and its variability in postmenopausal women with treated hypertension. Menopause 7 (1), 31±35. Van Der Vliet, A., Smith, D., O'Neill, C.A., Kaur, H., DarleyUsmar, V., Cross, C.E., Halliwell, B., 1994. Interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion. Biochem. J. 303, 295±301. Wieland, E., Parthasarathy, S., Steinberg, D., 1993. Peroxidase dependent metal-independent oxidation of low density lipoprotein in vitro: a model for in vivo oxidation?. Proc. Natl Acad. Sci. USA 90, 5929±5933. Yamada, M., Yoshida, M., Hashinaka, K., 1993. Identi®cation of transcriptional cis-element in introns 7 and 9 of the myeloperoxidase gene. J. Biol. Chem. 268, 13,479±13,485. Zuckerman, S., Bryan, N., 1996. Inhibition of LDL oxidation and myeloperoxidase dependent tyrosyl radical formation by selective estrogen receptor modulator raloxifene /LY139481 HCL/. Atherosclerosis 126, 65±75.