Effects of development and ageing on pulmonary NADPH-cytochrome c reductase, glutathione peroxidase, glutathione reductase and thioredoxin reductase activities in male and female rats

Mechanisms o f Ageing and Development, 37 ( 1987) 183 - 195

183

Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF DEVELOPMENT AND AGEING ON PULMONARY NADPHCYTOCHROME c REDUCTASE, GLUTATHIONE PEROXIDASE, GLUTATHIONE REDUCrASE AND THIOREDOXIN REDUCTASE ACTIVITIES IN MALE AND FEMALE RATS

CONSUELO SANTA MARIA and ALBERTO MACHADO De partamento de Bioq u imica, Facultad de Farmacia. Universidad de Sev illa. Sevilla fSpain/

(Received December 1lth, 1985) (Revision received September 26th, 1986)

SUMMARY The behaviour of the principal NADPH-consuming detoxification enzymes (NADPHcytochrome c reductase, glutathione peroxidase-glutathione reductase system, and thioredoxin reductase) was studied during .development and senescence of the rat lung. We have also studied the influence of sex on the development and senescent values. The NADPH-cytochrome c reductase activity increases at birth and afterwards remains constant until the 25th day after birth, at which age there is a maximum activity. Its activity decreases during the ageing period in both sexes. The glutathione reductase and thioredoxin reductase activities show significant differences with respect to sex during the adult stage, however during ageing these differences disappear. These enzymes show maximum activity at 25 days after birth, and afterwards the activity decreases continuously until the adult levels are reached. The activity of glutathione reductase is increased during the ageing period, especially in the female rats, however, in senescence the levels of thioredoxin reductase are lower than in the adult stage. The glutathione peroxidase shows a significant difference between both sexes during senescence and in the male its activity in this stage is higher than during development and adulthood.

Key words: Lung; Development and ageing rat; NADPH-cytochrome c reductase ; Thiore-

doxin reductase; Glutathione peroxidase; Glutathione reductase

Address all correspondence to: Alberto Machado, Departamento de Bioqulmica, Facultad de Faxmacia,

cfrramontana s/n, Sevilla41012, Spain. 0047-6374/87/$03.50 Printed and Published in Ireland

© 1987 Elsevier Scientific Publishers Ireland Ltd.

184 I NTRODU('TION Age and sex are two of the most important factors which are responsible for the variation in the animal detoxification capacity [1-5]. The foetus has a very low detoxification capacity, probably due to the placental barrier protection [6]. Since the initial studies of Fouts and Adamson [7], who found a very low hepatic microsomal mixed function, oxidase activity at birth, the development of the detoxification system has been extensively studied'in liver 18,9]. Furthermore, a number of clinical studies have reported age-dependent declines in the metabolic rates of various drugs and xenobiotics [10]. These clinical data are supported by in vitro and in vivo experimental studies which demonstrated marked age-related declines in the activities of liver drug-metabolizing enzymes in rats [11--14]. However, no similar attention has been paid to the development or ageing of these enzymes in extrahepatic tissues, although a variety of drug-metabolizing enzymes, such as arylhydrocarbon hydrolase, epoxide hydrolase, glutathione S-transferase and uridine diphosphoglucuronyl transferase has been detected in several extrahepatic tissues [15]. The lung is one of the extrahepatic tissues with the largest metabolic capacity for drugs [6], and in this organ the respiratory exposure to volatile compounds may present a greater risk than in other tissues. Taken together these findings lead us to undertake an investigation of the behaviour of the main NADPH-consuming detoxification enzymes (NADPH-cytochrome c reductase, glutathione peroxidase-glutathione reductase system, and thioredoxin reductase) during the development and senescence of the rat lung. We have also studied the influence of sex on their development and senescent profiles. At the same time, some of these enzymes have an additional interest in studies of the ageing period, taking into account that these are related to the enzyme elimination of some products of oxygen that have been suggested as being involved in the ageing process [16,17]. MATERIALS AND METttODS Chemicals

NADPH (nicotinamide adenine dinucleotide phosphate, tetrasodium salt Type I), cytochrome c (from horse heart salt free), EDTA (ethylenediaminetetraacetic acid, disodium salt), DTNB (5,5'-dithio bis 2-nitrobenzoic acid), DDT (DL-dithiothreitoi), glutathione reductase (EC 1.6.4.2), GSH (reduced glutathione), and GSSG (oxidized glutathione) were all purchased from Sigma Chemical Co. All reagents were of the highest purity available from commercial sources. Animals

Male and female Wistar rats of different ages (-4,0,5,10,15,20,25 and 30 days, and 3, 6 and 24 months) were used. They were sustained on a commercial stock diet throughout their life. Food and tap water were given ad libitum. None of the animals were found

185 to have gross pathological lesions. The rats were maintained at 21-22°C with 12-h light/ dark cycles.

Tissue preparations The animals were killed by cervical dislocation. To minimize possible diurnal variations the rats were routinely killed between 0900 and 1000 h. The lungs were immediately rinsed in ice-cold 0.145 M NaCI, trimmed and quickly weighed. All subsequent processing were carried out at 0-4°C. Homogenates (10% w/v) were prepared in 0.25 M sucrose, lmM EDTA, lmM DL-dithiothreitol and 15 mM Tris-HCl (pH 7.4) by using an allglass Potter Elvehjem homogenizer. The centrifugation was carried out at 800 g for 15 rain. The 800 g supernatant was used to determine enzyme activities. All samples were kept at 0°C until assayed.

Enzyme assays NADPH-cytochrome c reductase was measured as has been described by Vermilion and Coon [18]. The 1.0 ml assay mixture contained the following components: 300 mM phosphate buffer (pH 7.7), 0.04 mM cytochrome c, 0.1 mM EDTA, 0.2 mM NADPH and tissue (0.05-2 mg). The reaction was initiated by the addition of the NADPH, and the reduction of cytochrome c was followed spectrophotometrically at 550 nm. Glutathione peroxidase activity w~is assayed with a coupled system in which GSSG reduction was coupled to NADPH oxidation by glutathione reductase [19]. The assay mixture contained: 0.1 M potassium phosphate (pH 7.5), 5 mM EDTA, 2 mM NAN3, 1 mM GSH, 0.2 mM NADPH, 4/ag glutathione reductase and tissue sample (0.05-2 mg). After 10 rain preincubation (25°C) the reaction was initiated by the addition of 0.05 ml of 5.0 mM H20: (final vol., 1.0 ml). Glutathione reductase activity was determined spectrophotometrically by measuring NADPH oxidation at 340 nm [20]. The reaction mixture contained: 0.1 M potassium phosphate (pH 7.5), 5 mM EDTA, 4 mM GSSG and tissue (0.25-10 mg). The reaction was initiated by the addition of 0.1 ml of 2 mM NADPH. The rate of NADPH oxidation in the absence of GSSG was also determined and substracted from the overall rate. The thioredoxin reductase was measured as reported by Holmgrem [21 ]. The reaction mixture contained: 100 mM potassium phosphate (pH 7), 10 mM EDTA, bovine serum albumin 0.2 mg/ml, DTNB 5 mM and tissue ((0.25-10 mg). The reaction was initiated by the addition of 0.02 ml of NADPH 25 mM, and the reduction of DTNB was followed spectrophotometrically at 412 nm. All spectrophotometric measurements were carried out in a Pye-Unicam SP 1700 ultraviolet spectrophotometer, with 1.0 ml quartz cuvettes with a light path of 1.0 cm. All enzyme assays were realized at 25°C except glutathione reductase which was assayed at 37°C. Specific activities were expressed as nmol of substrate oxidized or reduced/min per mg of protein. Protein concentrations were determined by the method of Lowry et al. [22] with serum bovine albumin (Fraction V) as the standard.

1 86

Statistical methods Student's t-test was utilized to evaluated differences between means, and the 0.05 level of probability was used as the criterion of significance.

R I~S L: I.TS

NADPH-cytochrome c reductase NADPH-cytochrome c reductase is an important constituent of the structural portions of membranes, and its enzymatic functions allow the hydroxylation of a great variety of xenobiotics [23,24]. Fig. 1 represents the developmental and ageing profiles of this enzymatic activity in the lung for both sexes of the rat. As it can be seen, the activity of this enzyme does not show any sex-dependent effect in the lung. This is in agreement with the behaviour of

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187

hepatic NADPH-cytochrome c reductase [25]. However, Kitagawa et al. [26] found sexdependence in adult hepatic enzyme (3 months), although no differences were found at other ages o f the rat. The NADPH-cytochrome c reductase activity increases at birth, and remains more or less steady until 25 days after birth, this being when the maximum activity is reached. The liver enzyme, also reaches its maximum activity around this period [27,28]. From 25 days onwards, NADPH-cytochrome c reductase activity decreases continuously, and the activity observed in 3 month old rats was similar to those found on the fourth day before birth. The adult lung activity is 30-fold lower than in the liver and 4-fold lower than in the brain [24]. In the ageing period, we have found a progressive loss of NADPH-cytochrome c reductase activity. The activity found in 24 month old rats is about 5 and 2.5 lower in male and female rats respectively to those found in adulthood.

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Fig. 2. Specific activity of glutathione peroxidase in lung of female (.) and male (e) rats during development and ageing. Activities are expressed in nmol • mg protein -t • rain -~ . The results are means ± S.E.M. for five experiments. Statistical significance: *P < 0.001 (difference between sexes). Abbreviations: T, term; A, adult (90 days).

188

Glutathione peroxidase-gtutathione reductase system The enzymes glutathione peroxidase and glutathione reductase are implicated in the oxidation and reduction of glutathione and detoxification of peroxides [29]. Glutathione peroxidase transforms hydrogen peroxide and lipid peroxides to their corresponding alcohols, at the same time, the GSH is oxidized to GSSG. To complete the oxidationreduction cycle, the glutathione reductase regenerates GSH from GSSG by utilizing NADPH. Figure 2 shows the pattern of variation of glutathione peroxidase activity in the lungs of male and female rats during life. As can be observed, its activity increases at birth, and the profiles during development are parallel in both sexes. During this time, the activity is increased about 2 - 3 times from the activity at day 4 before birth. However, from adult to 24 months there is a sex-dependent variation of its activity. Thus the maximum activity in males is found at 6 months and remains more t,, less constant until 24 months, while in females, the activity drops markedly from 3 months (maximum activity) onwards.

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189 These differences do not correspond with the sexual maturity, because in any case they have been visible before (between 1 and 3 months, when the rat reaches adult maturation). The development patterns of glutathione reductase in lungs of male and female rats are shown in Fig. 3. The activity and developmental profiles ofglutathione reductase show significant differences between the sexes up to 6 months of age, but these differences disappear during the ageing period. During development the maximum increase in activity is observed in the interval between birth and 25 days postnatal, when the sex differences are established. From then onwards, glutathione reductase activity decreases continuously until 3 months (adult stage). In the male, adults activity is around 1.5 fold higher than that found at birth, and the female is about 60% lower than that found at birth. During ageing the sex-dependent activity is lost. An increase in glutathione reductase activity, especially in the female, has been observed.

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Fig. 4. Specific activity of thioredoxin reductase in lung of female (.) and male (*) rats during development and ~eing. Activities are expressed in nmol •mg protein -I • min -~ . The results are means ± S.E.M. for 5 experiments. Statistical significance: *P < 0.005 (difference between sexes). Abbreviation s: T, term; A, adult (90 days).

190 Thk~redoxin reductase

As glutathione, thioredoxin seems to have a general protective role in protein SH groups [30]. Thioredoxin was first discovered as a donor of reducing equivalents for the ribonucleotide reductase reaction [31 ]. The thioredoxin reductase is necessary to maintain thioredoxin in its reduced form [32]. The thioredoxin system is able to reverse disulphide bond formation in some cortical proteins of rabbit reticulocyte lysates. As the concentrations of these proteins are in vivo 10/aM for thioredoxin and 2/aM for thioredoxin reductase, this system could produce a substantial reduction of disulphides of small molecular weight components and polipeptides [30]. Figure 4 shows the profiles of this enzyme for female and male rats throughout their life. The activities and developmental pattern of thioredoxin reductase for rat lung do not show any sex difference up to 3 months although the adult levels are significantly different in both sexes. The variations of this activity during development fall into two different phases, an increase, which takes place from days 4 to !0 postnatal, and a continuous decrease from then onwards. Since the decrease with time is higher in females than in males a significant difference (P < 0.005) between both sexes is observed in adults. The activity in adult males is only slightly lower than that at birth, whereas the adult female has about 3-fold lower activity than the newborn one. The sex-dependent differences in thioredoxin reductase activity, found in adulthood, disappear during ageing. In this period, the decrease in enzymatic activity of the male rat lung is more pronounced than in the female. Similar and very low levels of thioredoxin reductase in the lung of old rats of both sexes are found. DISCUSSION Many theories have been proposed for the ageing process. Among other possibilities, it has been suggested that ageing tissues become more susceptible to oxidative tissue damage produced by free radicals [33]. Dietary manipulations expected to lower the rate of production of free radical reaction damage have been shown to increase the lifespan of mice, rats, flies and others [34,35]. Another important fact is that senescent organisms are probably less effective in metabolizing xenobiotics, including therapeutic drugs, which exhibit their toxicity by inducing membrane lipid peroxidation [36]. It is known that an increase in the susceptibility to drugs and foreign compounds occurs in conjugation with the ageing process, and a decrease in the ability to metabolize drugs may, in part, account for this phenomenon [Ill. One of the extrahepatic tissues with a great detoxification capacity for drugs is the lung [6]. In addition to being a point of entry into the body of airborne material, be it drugs or environmental contaminants, the lungs have the ability to concentrate many basic drugs irrespective of their route of administration [6]. Several oxidations mediated by mixed function oxidases (MFO) have been detected in the lung [6]. NADPH-cytochrome c reductase is an important component of this MFO. This enzyme allows the hydroxylation of a wide variety of endogenous and

191 exogenous substrates: steroids, fatty acids, prostaglandins, drugs and environmental chemicals [37]. The few activities of this enzyme that we have found in the lung in comparison with other tissues [24] suggest that the lung has little hydroxylation capacity. However, postnatal increase of this activity, similar to that found in the liver, could respond to the sudden restoration of ventilation along with a simultaneous exposure to airborne xenobiotics and related compounds. At the same time, the early drop of this activity could be related to the decline in survival of 96-98% oxygen exposure that starts around 35 days [38]. In the ageing period we have found a progressive loss ofNADPH-cytochrome c reductase activity similar to that observed in the liver [2,3]. This behaviour is parallel in both sexes. The activity found in 24 months is about 5 times lower than that found in adulthood. Schmucker and Wang [39] have reported that the decrease in the specific activity of this enzyme in the liver of old rats is accompanied by changes in molecular characteristics of the enzyme, probably due to post-translational modifications. Similar processes could also be involved in the decrease of NADPH-cytochrome c reductase activity of the lungs during ageing. It is interesting to point out the lack of sex difference in the behaviour of this enzyme during development and ageing. Some authors have described sex differences in its induction by some compounds, e.g. phenobarbital produces a major increase of this enzyme in males, however, contrary results have been reported when spironolactone has been used [25]. The glutathione has an important function in the protection of enzyme oxidation by free radicals and also in some detoxification processes [40]. This tripeptide is present in all living cells in different intracellular levels that depend on the growth state, nutritional state, hormonal balance, etc. [41]. The lung has almost 5 times lower concentration of glutathione than the liver [42]. This proportion may be an important factor in the relative susceptibility of the lungs to the toxic and carcinogenic effects of xenobiotics and their metabolites, and also could affect other cellular functions, e.g. the reduction of thiol groups of proteins, the redox state, and the activity of the same enzyme utilizing glutathione as cofactor or substrate. Moreover, it has been reported that the GSH concentration was decreased during ageing in many tissues and also in the lung [43]. It has been suggested that an enhanced oxidation via the glutathione peroxidase-glutathione reductase cycle was a possible explanation for the ageing decline in the ageing [44]. The glutathione peroxidase levels, found by us, in the foetal stage is significantly lower than in adulthood (Fig. 2). These results could be corroborated by the low levels of this enzyme in foetal animals [45]. The ageing pattern of this enzyme in the male is similar to that observed in the heart [46] and brain mitochondria [47] when the enzyme undergoes a significant age-dependent increase. Similar results have been reported by Kitahara [3]. This pattern is also similar to that reported by Spearman and Leibman [48] in the activity of glutathione S-transferase, a functionally-related glutathione metabolizing enzyme, in the rat lung, liver and brain. However, contrary to this behaviour of glutathione peroxidase in the lung.

192 Hazelton and Lang [44] have reported a decrease in this liver activity in old males. These results could be in accordance with the knowledge that the males have a higher drugmetabolizing capacity [49]. Generally, male rats are more resistant to the effects of several drugs than female rats. It seems to be due to an androgenic effect, demonstrated by the fact that the castration of male rats decreases the rate of metabolism of some drugs, while the administration of testosterone to female rats enhances their metabolism [91. The action of glutathione peroxidase in reducing hydroperoxides is dependent on the availability of reduced glutathione which is, in turn, maintained by the novo synthesis via glutathione synthetase and glutathione reductase activities. Glutathione reductase in lung was found to be only about 20% of that in the liver [42]. This activity is sufficient to keep virtually all of the lung glutathione reduced under normal conditions. Therefore, the liver may be able to cope with the rapid oxidation of glutathione (e.g. large amounts of H202 in the presence of glutathione peroxidase) better than the lung. We have found that the developmental pattern of this enzyme is similar to those of several enzymes involved in lipid biosynthesis [50] including the adaptative enzymes reducing NADP. In adulthood, the male animals show higher activity than female ones. The same results have been reported for liver by igarashi [51] who also found significant differences in hepatic intracellular GSH but not in CSSG levels. This fact may be explained, at least in part, by the higher amount of extracellularly transported GSSG in females than in males [52]. During ageing, the activity of this enzyme changes little in male animal lungs. Similar results have been reported by Kitahara e t al. [3] in the liver, but differ with the studies reported by Hazelton and Lang [44] in the mouse liver. The thioredoxin reductase is an enzyme with a different activity in both sexes, about twice in male than in female (Fig. 4). The reason for this sex difference would be similar to that observed for glutathione reductase and peroxidase, which could be related to estrogen levels and pituitary factors as it has been proposed for a large group of liver enzymes [26,53]. The sex-dependent decrease in the female lung enzyme activity could be therefore related to differences in protein synthetizing rates between both sexes. At the same time, many other functions have been proposed for the thioredoxin and thioredoxin reductase, some of which could be responsible for this sex difference. For example, the thioredoxin is the endogenous glucocorticoid receptor activation factor [54] although the mechanism of action of thioredoxin (reduction of disulfide bonds or high-affinity binding to the receptor) remains to be established. It can also be observed that the sex-dependent differences in thioredoxin reductase activity disappear during ageing. Similar and very low levels of thioredoxin reductase in the lung of old rats in both sexes are found. Taking into account some of the biological functions of thioredoxin reductase, the decrease of its activity could have important metabolic implications. In summary, this study demonstrates that, as have been described for the liver, some drug-metabolizing enzymes in the lung change according to we and sex. This could

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