Effect of age and photoperiodic conditions on

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significant modification was observed in CYP1A- and 2E-dependent enzyme activities ... In the first (basic) experiment 6 groups of 5/6 animals of each age were ...
Life Sciences 73 (2003) 327 – 335 www.elsevier.com/locate/lifescie

Effect of age and photoperiodic conditions on metabolism and oxidative stress related markers at different circadian stages in rat liver and kidney C. Martin a, H. Dutertre-Catella a,b,*, M. Radionoff a, M. Debray c, C. Benstaali d, P. Rat a, M. Thevenin a, Y. Touitou d, J.-M. Warnet a a

Laboratoire de Toxicologie, Faculte´ de Pharmacie, Universite´ Paris V, 4 Avenue de l’Observatoire, 75006 Paris, France b Laboratoire de Toxicologie, Faculte´ de Pharmacie, Universite´ Francßois-Rabelais de Tours, 31 Avenue Monge, 37200 Tours, France c Laboratoire de Mathe´matiques, Faculte´ de Pharmacie, Universite´ Paris V, 4 Avenue de l’Observatoire, 75006 Paris, France d Laboratoire de Biochimie me´dicale et Biologie mole´culaire, Centre Hospitalier Pitie´-Salpeˆtrie`re, 91 Boulevard de l’Hoˆpital, 75013 Paris, France Received 27 August 2002; accepted 7 January 2003

Abstract It has been shown that some cytochrome P450-dependent enzyme activities could present daily fluctuations, particularly CYP3A isoenzymes which are enhanced during the dark period. The aim of this study was to investigate whether age and photoperiodic conditions at different circadian stages could influence these fluctuations. Young mature (10 weeks) and old (22 months) Wistar rats were initially exposed to light-dark cycles 12:12 during 4 weeks, and secondly 18:6 for either one week or six weeks. Erythromycin N-demethylase (CYP3Adependent), 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydroxylase (CYP2E-dependent) activities were determined in liver and kidney microsomes at different hours after darkness onset (HADO). In addition, liver and kidney GSH, GSHPx, ATP, TBARS were determined. During the LD 12:12 cycle, while no significant modification was observed in CYP1A- and 2E-dependent enzyme activities as functions of HADO, erythromycin N-demethylase activity (CYP3A-dependent) showed a significant increase during the second third of the dark period in both young and old rats. After switching to a LD 18:6 cycle, this variation was still observed during second third of the dark period, to a lesser but still significant degree, with no difference between one week and six weeks exposure to the new photoperiod. It can be noted that the old rats showed a significantly lower level

* Corresponding author. Laboratoire de Toxicologie, Faculte´ de Pharmacie, 4 Avenue de l’Observatoire, 75006 Paris, France. Tel.: +33-153-73-98-64; fax: +33-143-26-71-22. E-mail address: [email protected] (H. Dutertre-Catella). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00271-6

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of erythromycin N-demethylase activity than the young rats, in parallel to a decrease in GSH, GSHPx and ATP, and an increase in TBARS. These results confirm the lower resistance of old animals to oxidative stress. The observed variations in metabolism parameters underline the need for study designs in pharmaco-toxicology taking into account the possible risks induced by circadian changes, especially in aged subjects. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Circadian rhythm; Photoperiod; Age; Metabolism markers; Oxidative stress

Introduction Aging is often considered to be related to a loss of the temporal structure with a decrease of the adaptation capability of various functions of the organism (Touitou and Haus, 2000). Age-related modifications in the parameters of circadian rhythms include decreased amplitude, modifications in the phase and reduced sensitivity of the circadian pacemaker to photic and non-photic signals (Mailloux et al., 1999; Touitou and Haus, 1994). These alterations can be either the cause or the consequence of modifications in the biological clock which adjusts (entrains) the circadian function with the alternation of light and dark, the most important synchronizing agent for the rat circadian system. Besides, rhythm modifications may be involved in damage processes of cells and organs. It is well known that drug efficacy and/or toxicity undergo daily variations which have been attributed to daily fluctuations in liver drug-metabolizing enzyme activities (Belanger, 1996). Recently, Furukawa et al. investigated various liver cytochrome P450 isoenzyme activities, finding high values during the dark period and low values during the light period, both in fasted and non-fasted male rats (Furukawa et al., 1999). This suggests that fluctuations of P450-dependent enzyme activities might not be related to the quantitative variation of P450 proteins. The majority of these studies have been carried out in either young growing or young mature rats. Taking into account this background we found worth investigating the effects of age on different liver and kidney metabolism markers under a standard lighting condition (LD 12:12) then the adaptation of these rhythms to a long photoperiod (LD 18:6).

Materials and methods Animals The animal experimentation was conducted in accordance with university and national guidelines. Male Wistar rats aged 10 weeks (young mature) or 22 months (old) purchased from Janvier (Le Genest Saint-Isle - France) were housed one per cage with free access to standard rat chow and tap water. They were placed in chronobiologic animal facilities (ref. E110-SP-6, A110-SP, ESI FluFrance, Arcueil, France), under artificial lighting conditions of 12 hours of light (L) and 12 hours of dark (D) per day (LD 12:12). The chronobiologic facilities were sound proof, temperature-controlled (23 F 1.0 jC), provided with filtered air. Each compartment had its own lighting control, allowing to obtain different chronobiological cycles in each compartment. Light was provided by 40 W fluorescent tubes, with an intensity of 50 lux in the middle of the animal cage. Litter, water and food were changed once a week, during the

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dark span, conducted under a red light to avoid phase-shift of the circadian rhythms and disturbances during the rest phase. Experimental protocol In the first (basic) experiment 6 groups of 5/6 animals of each age were maintained on a LD 12:12 cycle for four weeks. In the second experiment the animals were first acclimatized under the same light/ dark conditions for four weeks, then submitted to a LD 18:6 cycle for one week (6 groups of each age) or six weeks (6 groups of each age). At the end of the 1st experiment (LD 12:12), the rats were sacrificed on the same day by decapitation at the following times: 0 hours after darkness onset (HADO); 4 HADO corresponding to 1/3 of the dark period (DP); 7 HADO corresponding to approximately 2/3 DP; 12 HADO corresponding to 3/3 DP; 18 HADO corresponding to 1/2 of the light period (LP) and 20 HADO, corresponding to approximately 2/3 LP (Fig. 1a). At the end of the 2nd experiment (LD 12:12 4 weeks + LD 18:6 one week or 6 weeks), the rats were sacrificed on the same day by decapitation at the following times at the same relative intervals of DP and LP: 0 HADO; 2 HADO (1/3 DP); 3.5 HADO ( c 2/3DP); 6 HADO (3/3 DP); 15 HADO (1/2 LP); 22 HADO ( c 2/3 LP) (Fig. 1b).

Fig. 1. Experimental procedure.

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Tissue preparation The liver and kidneys were immediately removed, rinsed with cold isotonic NaCl, frozen in liquid nitrogen and stored at 80 jC until used. The thawed liver and kidney samples were homogenized in TRIS-KCl buffer at pH = 7.5. Cytosol and microsomal pellets were prepared in TRIS-KCl buffer at pH = 7.5 by ultracentrifugation (Thomas et al., 1983). Analytical methods The protein content of the homogenate, cytosol and microsomes was measured using the Pierce BCA protein assay kit with serum bovine albumin as standard. Glutathione concentration was determined in the homogenate by spectrophotometry (Griffith, 1980), and glutathione peroxidase activity in the cytosol by cumene hydroperoxide reaction (Wendel, 1981). ATP concentration was measured in the homogenate by enzymatic determination (Adams, 1963). Lipid peroxidation was assessed by the amount of malondialdehyde formed in the presence of the microsomal pellets (Malvy et al., 1980). The total cytochrome P450 content in the microsomal fraction was measured by the method of Omura and Sato (Omura and Sato,

Fig. 2. Daily fluctuations of liver erythromycin N-demethylase activity before (2a) and after (2b) desynchronization (n = 5/6 rats per group).

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1964). Erythromycin N-demethylase,7-ethroxycoumarin O-deethylase and aniline 4-hydroxylase activities were determined in the microsomal fraction respectively by the methods of Nash (1953), Mazel (1971) and Ullrich and Weber (1972). Statistical analysis The results are expressed as mean F S.D. The daily fluctuation of all parameters was estimated by analysis of variance ANOVA, followed by Dunnett’s test (p < 0.05). Comparisons between young and old rats at each HADO were carried out using Student’s t test with Bonferroni’s adjustment (p < 0.05).

Results Metabolism markers Liver Liver erythromycin N-demethylase (CYP3A-dependent) activity showed an important and significant increase during the second third of the dark period in both young (# 90%) and old (# 35%) rats during

Fig. 3. Liver erythromycin N-demethylase activity (CYP3A-dependent) (n = 5/6 rats per group). SD not shown here to avoid overloading of figure 3 (for LD 12:12 4 weeks see Fig. 2a, for LD 12:12 4 weeks + LD 18:6 1 week see Fig. 2b, for LD 12:12 4 weeks + LD 18:6 6 weeks, data not shown, no significant difference with LD 18:6 1 week).

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Table 1 Oxidative stress related markers Liver GSH AM/g organ GSH-PX AM/min/mg protein ATP AM/mg protein TBARS AM/mg protein

Kidney

Old

Young

ratio Old/young

Old

Young

ratio Old/young

9.25 0.56 1.35 2.15

11.54 1.35 1.66 1.40

0.80*** 0.41*** 0.81** 1.54**

1.06 0.78 1.60 1.66

1.23 1.23 1.78 1.35

0.86** 0.63** 0.90* 1.23*

* p < 0.05. ** p < 0.01. *** p < 0.001.

the LD 12:12 cycle. However, the increase observed with young rats was about 2.5 times that observed with old rats (Fig. 2a). The same kind of variation was found during the second third of the dark period to a lesser but still significant degree, both after one week and six weeks of exposure to a new photoperiod (LD 18:6). The increase observed in the young rats was approximately 40%, and twice that observed in the old rats (Fig. 2b). By contrast, no significant modifications in 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydrolase (CYP2E-dependent) activities were observed. Fig. 3 underlines that these increases in liver erythromycin N-demethylase activity occur in all cases at the second third of the dark period. The difference in amplitude between LD 12:12 4 weeks and LD 12:12 4 weeks + LD 18:6 1 week is respectively 18.5% in young rats and 8.7% in old rats but none of these values are significant. Kidney In the kidney, no increase in the three enzyme activities was found after exposure to any of the different photoperiods, LD 12:12 or LD 18:6.

Fig. 4. GSH-Px (AM/min/mg protein).

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Oxidative stress related markers Significant decreases in GSH, GSH peroxidase and ATP, together with a significant increase in lipid peroxidation (TBARS) were observed in the old rats compared to the young rats, both in the liver and in the kidney (Table 1). No difference in any of these markers was observed between LD 12:12 and LD 18:6 cycles whatever the HADO, thus the comparisons were made between pooled data of the young rats vs pooled data of the old ones (Fig. 4: ex: GSH-Px).

Discussion A well-established basis of the temporal structure of liver and kidney is important for a sound approach of the biological functions of these organs. It has been previously shown that the activities of hepatic and kidney enzymes are not constant during a 24h period (Touitou and Haus, 1994). Most cells are capable of expressing circadian variations driven by oscillators (or pacemakers) in which rhythmicity is entrained by environmental factors such as the light-dark cycle, among others (Touitou and Haus, 1994). We document in this paper the photoperiod-dependent variations on liver and kidney cytochrome P450-dependent enzyme activities: CYP3A-, CYP1A- and CYP2E-dependent enzymes. It is the first time, to our knowledge, that such effects have been studied. We also looked at markers of oxidative stress. This research was carried out within and between groups of young mature and old Wistar rats. When animals were on a LD 12:12 cycle, we found an important and significant increase in liver erythromycin N-demethylase activity (CYP3A-dependent) during the 2nd third of the dark period both in young mature and old rats (respectively 90% and 35%), but not in 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and in aniline 4-hydroxylase (CYP2E-dependent) activities. This difference could be explained by the fact that CYP3A is quantitatively the most important protein involved in drug metabolism and that our study has been carried out without previous enzyme-inducing treatment. Although Furukawa et al. (1999) found circadian variations with the 3 enzyme liver activities they tested in Fischer rats: 7-methoxy-, ethoxy- and propoxy-coumarin O-dealkylases, respectively 2B-, 1A- and 3A-dependent, this divergence with our results could be due to rat strain, age or to a different specificity of the substrates. When animals were on a LD 18:6 cycle, the increase in erythromycin N-demethylase activity also occurred during the 2nd third of the dark period, both after 1 week and 6 weeks. This shows that, after 1 week, the animals were already adapted to the new LD cycle. The lower increase observed in the old rats, after exposure to both types of LD cycles, is probably due to decreased basal levels of CYPs as it is known that CYP inducibility decreases with age (Lee and Werlin, 1995). No daily variations in enzyme activities were found in the kidney. As P450 protein quantities are 8 to 10 times lower in the kidney than in the liver and thus enzyme-related activities are very low, it is not surprising that no data were found in the literature. Further research on the subject is therefore needed. Some authors have found circadian variations in liver glutathione concentration or in lipid peroxidation in adult rats but with conflicting results concerning the period of high and low values (Farooqui and Ahmed, 1984; Belanger et al., 1991; Tun˜on et al., 1992). Tun˜on has suggested that

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periodical feeding can influence the level of liver enzyme activities (‘‘masking effects’’) by means of a modification of circadian rhythm parameters. Under our experimental conditions, no circadian variations in oxidative stress related markers were observed in the liver and the kidney either in young or old rats. By contrast, we have shown parallel significant decreases in oxidative stress related markers in these two organs, particularly an important decrease in GSH-Px activity in old rats, demonstrating that the capacity to maintain a balance of the redox intracellular state is not well preserved in the old age. Conflicting results have been published regarding the relation between lipid peroxidation and antioxidative enzyme activities during the aging process, probably due to species-, strain-, gender- or tissue-specific differences (Rikans and Hornbrook, 1997). In conclusion, these time-dependent variations are important from a clinical point of view because they are involved in changes around the clock in e.g. pharmacokinetics, pharmacodynamics, and drug toxicity. It is well known that aged subjects are more susceptible to hepatic and renal adverse effects of xenobiotics. Our results confirm the lower resistance of old animals to oxidative stress. Since the liver and the kidney are the main target organs of xenobiotics, our results suggest the interest of considering metabolism parameters and oxidative stress status of these organs as a function of age in the design of long-term toxicology studies. Acknowledgements We wish to thank the ‘‘Association pour le De´veloppement de la Recherche en Toxicologie Expe´rimentale’’ (ADRTE) for financial support. References Adams, H., 1963. Adenosine 5V-triphosphate determination with phosphoglycerate kinase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, pp. 539 – 543. Belanger, P.M., Desgagne´, M., Bruguerolle, B., 1991. Temporal variations in microsomal lipid peroxidation and in glutathione concentration of rat liver. Drug Metabolism and Disposition 19 (1), 241 – 244. Belanger, P.M., 1996. Circadian rhythms in hepatic biotransformation of drugs. Pathologie Biologie 44 (6), 564 – 570. Farooqui, M.Y.H., Ahmed, A.E., 1984. Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats. Life Sciences 34 (24), 2413 – 2418. Furukawa, T., Manabe, S., Ohashi, Y., Sharyo, S., Kimura, K., Mori, Y., 1999. Daily fluctuation of 7-alkoxycoumarin Odealkylase activities in the liver of male F344 rats under ad libitum feeding or fasting condition. Toxicology Letters 108 (1), 11 – 16. Griffith, O.W., 1980. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Analytical Biochemistry 106 (1), 207 – 212. Lee, P.C., Werlin, S.L., 1995. The induction of hepatic cytochrome P450 3A in rats: effects of age. Proceedings of the Society for Experimental Biology and Medicine 210 (2), 134 – 139. Mailloux, A., Benstaali, C., Bogdan, A., Auzeby, A., Touitou, Y., 1999. Body temperature and locomotor activity as marker rhythms of aging of the circadian system in rodents. Experimental Gerontology 34 (6), 733 – 740. Malvy, C., Paoletti, C., Searle, A.J.F., Willson, R.L., 1980. Lipid peroxidation in liver: hydroxydimethylcarbazole a new potent inhibitor. Biochemical and Biophysical Research Communications 95 (2), 734 – 737. Mazel, P., 1971. Experiments illustrating drug metabolism in vitro. In: La Du, B.N., Mandel, H.G., Way, E.L. (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, pp. 546. Nash, T., 1953. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochemical Journal 55, 416 – 421.

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