Archives Internationales de Physiologie, de Biochimie el de Biophysique, 1992, 100, 83-87
83
Requ le 25 avril 1991.
Circadian rhythms in glutathione and glutathione-S transferase activity of rat liver BY
M. J. T U R 6 N ('), P. GONZALEZ (I), P. L6PEZ G. M. SALIDO (*) and J. A. MADRID (7 [('I Department of Physiology, Pharmacology and Toxicology, University of Leon; v) Department of Physiology, University of Extremadura; (9Department of Physiology and Pharmacology, Faculty of Biology, University of Murcia, Spain]. (l),
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
(3 figures)
The experiments were conduced to examine the existence of circadian rhythms in glutathione concentration and glutathione S-transferase activity in the liver of the rat. In animals synchronized to a 12:12 h light-dark cycle and fasted at 6 different time points to allow exactly 24 h of fasting, both, glutathione concentration and glutathione S-transferase activity show diurnal variation with a maximum during the light period and a minimum at night. On the other hand the hepatic protein level was maximal during the light period and decreased to its lowest level during the dark period. The implications of such oscillations in the circadian rhuthms of toxicological or therapeutical effects of many xenobiotic agents are clear.
Introduction Glutathione (L-y-glutamyl-L-cysteinglycine) is the most abundant thiol in virtually all mammalian cells. Glutathione performs a variety of physiological and metabolic functions. These include thiol transfer reactions which seem to protect cell membranes and proteins, metabolism of endogenous compounds and & ANDERSON, 1983). transport of aminoacids (MEISTER One of its most important functions is detoxification of drugs and xenobiotics by means of glutathione Stransferases (GST). These enzymes catalize the reaction between the nucleophile reduced glutathione and a large number of electrophilic compounds (BOYER, 1989; KETTERER,1986). The liver, having the greatest content of glutathione, is the major organ involved in the elimination and detoxification of xenobiotics and it also seems to play a central role in the interorgan homeostasis of et al., 1985). Studies by difglutathione (KAPLOWITZ ferent authors have found pronounced diurnal variations in the hepatic concentrations of a number of metabolic substrates and in the activity of different liver enzymes in the rat (BHATTACHARYA, 1981; HARDELAND et al., 1973; HOPKINS et al., 1973; VANNOORDEN et al., 1984). Although there are a number of studies reporting a day-night rhythm in liver concentrations of reduced and oxidized glutathione in rats (BECKet al., 1958; FARROOQUI & AHMED, 1984; ISAACS & BINKLEY, & PFAFF,1985; PREIN1977; LESZYNSKA-BISSWANGER INGER et al., 1961)there is still some disagreement upon the diurnal periodicity of hepatic glutathione concentration. Additionally, although diurnal variations for some glutathione enzyme-related have been described in rat brain (Dm-MVr;roz et al., 1985) and mouse liver
(NORTH et al., 1981), no information exists on the possible diurnal changes of GST activity. The purpose of the present study was to investigate in 24 h fasted animals the existence of circadian rhythms of glutathione concentration and GST activity in rat liver using a computerized inferential statistical method. Material and Methods
Reagents Bovine serum albumin, l-chloro-2,4-dinitrobenzoic acid, 5-5-dithiobis-(2-nitrobenzoicacid), reduced glutathione, glutathione reductase and nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest quality available. Distilled deionizad water was used throughout.
Animals and experimental procedure Thirty six adult male Wistar rats (Charles River, Barcelona, Spain) weighing 322.8 f 8.3 g, were maintained for three weeks under constant environmental temperature (21 f 1°C) and relative humidity (60%). The rats were exposed to a 12 h/12 h darkllight cycle (light on at 08 h) throughout an acclimatation period of 21 days. On experimental days (May 1990), the animals were fasted at six different time points, chosen as follows : 08, 12, 16, 20, 0 and 04 h. Exactly 24 h after fasting the animals were sacrificed by decapitation (6 animals were killed at each time) and the livers were carefully removed, weighed and immediately homogenized.
84
M. J. TURON, P . G O N Z ~ E Z ,P. LOPEZ, G. M. SALIDO AND J. A. MADRID
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
110
F 70
8
12
16
20
0
4
8
TIME (H)
FIG. 1. Circadian rhythm of rat liver protein. Time points values (top) are given as mean f SEM of six rats; calculated cosine curve of circadian variations is superimposed on the chronogram. Cosinor plot is shown at the botton. The circadian rhythm is represented by one vector originating at center of plot (pole). Length of vector represents rhythm’s amplitude. Vector direction indicates rhythm’s acrophase (highest point in rhythm). Circular region at tip of vector portrays joint confidence region (95%) for amplitude and acrophase. This region does not overlap the pole indicating a significant circadian rhythm. Arc described by tangents to region depicts conservative confidence interval for acrophase.
A nalitical procedures Determination of total glutathione content of liver prepared in 5q0 (w’v) thrichloroacetic acid in 0.01 N HCL was carried out as described by TIETZE (1969) with the modification of (1980). Hepatic cytOsO1ic glutathione stransferase activity was measured spectrophotometrically with 1-chloro-2, 4-dinitrobenzoic acid as substrate according to the methods of HABIG et al. (1974). Liver protein concentration was measured by the method Of et (1951) with bovine serum albumin as standard.
Statistical analysis All data were analysed for time-effects by the two ways analysis of variance (ANOVA) and for circadian
rhythm by a computerized inferential statistical et al., 1979),inmethod, the “single cosinor” (NELSON volving the fit of a 24 h cosine curve to averaged data series by the method of least squares. This method objectively determines the following information : - A probability or P value, which indicates the significance of the fit of the cosine curve to the data. If the P value was 0.05 or less, the fluctuation of the variable studied was presumed to be sinusoidal and not at random. - Three rhythmic parameters and their dispersions : these were designated as the acrophase, being either the time or angle of the crest of the fitted cosine curve; mesor being the cosinor determined overall 24-h mean, and amplitude being the half of the total cosine excursion. Both amplitude and mesor were expressed in the original units of the variable analyzed. In
GLUTATHIONE AND
4.5
GST : CIRCADIAN
85
RHYTHMS
-c
z c r =
4.0
0
E
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
03
3
-
S+ ko
3
N=6
3.5
-
3.0 i-I
I
I
I
I
I
L
I
I
I
FIG.2 . Circadian rhythm of liver glutathione. Chronogram (top) and cosinor test (botton) of liver glutathione concentration measured in six rats per time point. (For explanation see the legend of Figure 1).
addition to the mesor, amplitude, and acrophase, the coeficients of variation characterizing the data were calculated. The phasing was expressed in hours calculated in reference to local midnight. Results Although all animals exhibit a similar body weight, the hepatosomatic index [(liver wt/body wt) x 1001 shows a significant diurnal variation. The mean values at six different time points were :2.69, 2.75,2.92,2.87, 2.99, and 3.00%. Maximal hepatosomatic relations occurs during the dark period. The daily rhythm of cytosolic liver protein, shown in Figure 1 is typical of most rodents kept under the light-dark cycle indicated. The protein concentrations started to decrease at noon, reaching the lowest level in the midnight. The single cosinor analysis reveals the existence of a statistically significant circadian rhythm
characterized by a mesor of 94.96 mg/g liver (95% CI between 90.34 and 99.59), an amplitude of 16.30 mg/g liver (95% CI between 6.49 and 19.57) and an acrophase at 1310 h (95% CI between 1012 and 1557). CV = 93.04%. The diurnal rhythm of liver glutathione concentration, expressed as pmol/g liver, is shown in Figure 2. The minimum occurs in the dark period, gradually increasing as the light period begins. The mesor value is 3.75 pmol/g liver (95% CI between 3.63 and 3.87). The amplitude is 0.47 pmol/g liver and the acrophase occurs at 10.50 h (95% CI between 0848 and 1235). CV = 96.24%. Two activities of GST (nmol of conjugated CDNB/min. g liver and nmol of conjugated CDNB/min. mg protein) over a 24-h period are shown in Figure 3A and 3B. The diurnal patterns for both activities were quite similar. The two activities decreased during the dark period reaching the lowest values
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
86
M. J. TUSION, P. G O N Z ~ E Z ,P . LOPEZ, G. M. SALIDO AND J. A. MADRID
240
200 L
160
3-
Y
120
I,
a
I
I
12
I
I
16
I
I
20
0
4
a
TIME lFi\ FIG.3 . Diurnal changes in glutathione S-transferase activity. A : expressedper g of liver. B : expressedper mg of protein. Each point represents the mean i SEM of six animals. Continuous fine represents the best fitting cosine curve.
between OOOO and 0400 h, and then quickly increased to the high levels of activity observed in the light period. In both cases, non significant circadian rhythm can be detected by cosinor method because the data of enzyme activities are non-sinusoidal; however, the differences observed between data collected at different hours can be attributed to non-sinusoidal circadian rhythms, since revealed statistical differences bethe two-ways ANOVA tween time points. The GST activity shows an acrophase at 1325 h (nmol CDNB/min. g liver, Fig. 3A)and at 1312 h (nmol CDNB/min. mg protein, Fig. 3B) that partially coincides with those obtained for glutathione concentration. Discussion
Data by different authors indicate that activity of some hepatic enzymes and liver concentration of difet al., 1973;NOVELLO et al., ferent molecules (HOPKINS 1969) as well as the excretion of different biliary conet al., 1990)are stituents (DUANEet al., 1979;NAKANO modulated by food ingestion and follows a circadian pattern in the rat. With respect to glutathione metabolism it has been indicated that glutathione
concentration of liver (FAROOQUI & AHMED,1984; LESZCYSZYNSKA-BISSWANGER & PFAFF, 1985;SCHNELL et al., 1984), stomach (BODY et al., 1979), kidney & AHMED, 1984) and heart (DOROSHOW, (FAROOQUI 1979)exhibits a statistically significant diurnal variation in different species. Most of these time-dependent studies have been carried out with animals having spontaneous ad libitum or experimentallymodified feed patterns. However, periodical feeding can influence directly ("masking effect") the level of liver enzyme activities or indirectly, acting as a synchronizingagent, by means of a modification of circadian rhythm parameters, such as acrophase. In our experimental conditions we minimize the "masking" effects of food because every animal was fasted exactly 24 h prior to sacrifice. In this situation, the hepatic protein content shows a circadian rhythm which coincides with data previously published (SOLER et al., 1988). This protein rhythm is oppossed to hepatosomatic index oscillation and probably could be a reflex of the important rhythm & KHANDELWAL, 1985). in glycogen content (ROESLER Glutathione concentration also shows a diurnal variation coinciding with that of protein. The maintenance of hepatocellular glutathione is a dynamic
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
GLUTATHIONE AND
GST : CIRCADIAN RHYTHMS
process with a steady-state cellular concentration achieved by a balance between the rate of synthesis, catalized by glutamyl-cysteine synthetase and glutathione synthetase, the rate of utilization through redox and alkylating reactions and the rate of glutathione export from hepatocytes (NAKANO, 1990). The first enzymatic step is rate limiting in the synthesis of glutathione and largely limited by the availability of the free cysteine pool (VIRA et al., 1978): The degradation of glutathione is mainly accounted for sinusoidal efflux and in a minor part by consumption through conjugation reactions or enzymatic breakdown et al., 1985). (KAPLOWITZ GST activity also shows statistically significant diurnal variations, with a minimum during the maximal activity period of nocturnal rodents. This enzyme is involved in the detoxification of a large number of xenobiotics, including hepatic toxins such as carcinogens, a number of endogenous compounds including prostaglandins, leukotrienes, organic hydroperoxides and steroids are substrates for the GST (BOYER,1989). Thus, this rhythm can play an important role in the pharmacokinetic, toxicity and therapeutic effects of a number of drugs. Also the coincidence between diurnal changes in glutathione and in GST suggest that this enzymatic pathway can contribute to the circadian rhythm in glutathione concentration. However the possibility of changes in cysteine availability or glutathione export change remains open and further experiments are required to evaluate its contribution to circadian rhythm in the hepatic concentration of glutathione. Acknowledgments. - The authors are indebted to Mr. DENHAM WISDOMfor the advice on the English language.
References BECK,L. V., RIECK,V. D. & DUNCAN,B. (1958) Proc. SOC. Exp. Biol. 97, 229-231. BHATTACHARVA, R. D. (1981) in : Biological rhythms in structure and function, in : COSTO, P. M., VONMAYERSBACH, H., ScmvING, L. E. & PAULY, J. E. New York. BODY,S. C., SMAME,H. A. & BODY,M. R. (1979) Science 205, 1010-1012. BOYER,T. D. (1989) Hepatology 9, 486-496.
87 DIAZ-MUROZ,M., HERNANDEZ-MUAOZ, R., SUAREZ,J. & CHACOYA DE SANCHEZ,V. (1985) Neuroscience 16, 859-863. DOROSHOW, J. H., LOCKER,G. Y., BALDINGER, J. & MYERS,C. E. (1979) Res. Commun.Chem. Pathol. Pharmacol. 26,285-295. DUANE,W. C., GJLLVERSTADT, M. L. & WIEGAND,D. M. (1979) Am. J. Physiol. 236, R175-RI76. FAROOQUI, M. Y. & AHMED, A. E. (1984) Life Sci. 34, 2413-2418. GRLFFITH, 0 . W. (1980) Analyt. Biochem. 106, 207-212. HABIG,W. H., PALIST, M. J. & JAKOBY,W. B. (1974) J. Biol. Chem. 249, 7130-7139. HARDELAND, R., HOHMA", D. & RENSWG,L. (1973) J. Interdiscipl. Cycle Res. 4, 89-118. HOPKINS,H. A., BONNEY,R. J., WALKER,P. R., YAGER,J. D. & POTTER, V. R. (1973) Advanc. Enzyme Regul. 11, 169-191. ISAACS,J. & BINKLEY,F. (1977) Biochim. Biophys. Acta497, 192-204. KAp~owrrz,N., AW, T. Y., OOKHTENS,M. (1985) Ann. Rev. Pharmacol. Toxicol. 25, 715-744. KETTERER,B. (1986) Xenobiotic 10, 957-973. LESZCZYNSKA-BISSWANGER, A. & PEAEF, E. (1985) Biochem. Pharmacol. 34, 1635-1638. LOWRY,0 . H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-272. MEISTER,A. & ANDERSON, M. E. (1983) Ann. Rev. Biochem. 92, 711-760. NAKANO,A., TIETZ, P. S. & LARUSSON,N. F. (1990) Am. J. Physiol. 258, G653-G659. NELSON,W., TONG,L. Y. & HALBERG, F. (1979) Chronobiologia6, 305-323. NORTH,C., FEUERS, R. J., SCHEVING, L. E., PAULY,J. E., TSN, T. H. & CMCIANO,D. A. (1981) Am. J. Anat. 162, 183-199. NOVELLO, F., GUMAA, J. A. & MCLEAN,P. (1%9) Biochem. J. 111, 713-725. PREININGER, V., STEPAN,V., SERCE,J., KOJECKI,Z. & CERNOCH, M. (1961) Physiol. Bohemosl. 20, 307-311. R. L. (1985) Int. J. Biochem. 17, ROESLER, W. J. & KHANDELWAL, 81-85. SCHNELL, R. C., BOZIGIAN,H. P., DAVIES, M. H., MERRICK,B. A., PARK,K. S. & MCMJLLAN,D. A. (1984) Pharmacology 29, 149-157. SOLER,G., BAUTISTA,J. M., MADRID,J. A. & Sumo, G. M. (1988) Chronobiologia 15, 205-212. TIETZE,R. (1969) Analyt. Biochem. 27, 505-555. VANNOORDEN, C. J. F., BHATIACHARYA, R. D., VOOELS,I. M. C. & FRONIK, G. (1984) Chronobiologia 11, 131-138. H. & KREBS, H. A. (1978) Biochem. J. 170, VIAA,J., REGINALD, 627-630. J. A. MADRID, Department of Physiology and Pharmacology Faculty of Biology, University of Murcia Campus Espinardo, E-30100 Murcia. Spain.
83
Requ le 25 avril 1991.
Circadian rhythms in glutathione and glutathione-S transferase activity of rat liver BY
M. J. T U R 6 N ('), P. GONZALEZ (I), P. L6PEZ G. M. SALIDO (*) and J. A. MADRID (7 [('I Department of Physiology, Pharmacology and Toxicology, University of Leon; v) Department of Physiology, University of Extremadura; (9Department of Physiology and Pharmacology, Faculty of Biology, University of Murcia, Spain]. (l),
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
(3 figures)
The experiments were conduced to examine the existence of circadian rhythms in glutathione concentration and glutathione S-transferase activity in the liver of the rat. In animals synchronized to a 12:12 h light-dark cycle and fasted at 6 different time points to allow exactly 24 h of fasting, both, glutathione concentration and glutathione S-transferase activity show diurnal variation with a maximum during the light period and a minimum at night. On the other hand the hepatic protein level was maximal during the light period and decreased to its lowest level during the dark period. The implications of such oscillations in the circadian rhuthms of toxicological or therapeutical effects of many xenobiotic agents are clear.
Introduction Glutathione (L-y-glutamyl-L-cysteinglycine) is the most abundant thiol in virtually all mammalian cells. Glutathione performs a variety of physiological and metabolic functions. These include thiol transfer reactions which seem to protect cell membranes and proteins, metabolism of endogenous compounds and & ANDERSON, 1983). transport of aminoacids (MEISTER One of its most important functions is detoxification of drugs and xenobiotics by means of glutathione Stransferases (GST). These enzymes catalize the reaction between the nucleophile reduced glutathione and a large number of electrophilic compounds (BOYER, 1989; KETTERER,1986). The liver, having the greatest content of glutathione, is the major organ involved in the elimination and detoxification of xenobiotics and it also seems to play a central role in the interorgan homeostasis of et al., 1985). Studies by difglutathione (KAPLOWITZ ferent authors have found pronounced diurnal variations in the hepatic concentrations of a number of metabolic substrates and in the activity of different liver enzymes in the rat (BHATTACHARYA, 1981; HARDELAND et al., 1973; HOPKINS et al., 1973; VANNOORDEN et al., 1984). Although there are a number of studies reporting a day-night rhythm in liver concentrations of reduced and oxidized glutathione in rats (BECKet al., 1958; FARROOQUI & AHMED, 1984; ISAACS & BINKLEY, & PFAFF,1985; PREIN1977; LESZYNSKA-BISSWANGER INGER et al., 1961)there is still some disagreement upon the diurnal periodicity of hepatic glutathione concentration. Additionally, although diurnal variations for some glutathione enzyme-related have been described in rat brain (Dm-MVr;roz et al., 1985) and mouse liver
(NORTH et al., 1981), no information exists on the possible diurnal changes of GST activity. The purpose of the present study was to investigate in 24 h fasted animals the existence of circadian rhythms of glutathione concentration and GST activity in rat liver using a computerized inferential statistical method. Material and Methods
Reagents Bovine serum albumin, l-chloro-2,4-dinitrobenzoic acid, 5-5-dithiobis-(2-nitrobenzoicacid), reduced glutathione, glutathione reductase and nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest quality available. Distilled deionizad water was used throughout.
Animals and experimental procedure Thirty six adult male Wistar rats (Charles River, Barcelona, Spain) weighing 322.8 f 8.3 g, were maintained for three weeks under constant environmental temperature (21 f 1°C) and relative humidity (60%). The rats were exposed to a 12 h/12 h darkllight cycle (light on at 08 h) throughout an acclimatation period of 21 days. On experimental days (May 1990), the animals were fasted at six different time points, chosen as follows : 08, 12, 16, 20, 0 and 04 h. Exactly 24 h after fasting the animals were sacrificed by decapitation (6 animals were killed at each time) and the livers were carefully removed, weighed and immediately homogenized.
84
M. J. TURON, P . G O N Z ~ E Z ,P. LOPEZ, G. M. SALIDO AND J. A. MADRID
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
110
F 70
8
12
16
20
0
4
8
TIME (H)
FIG. 1. Circadian rhythm of rat liver protein. Time points values (top) are given as mean f SEM of six rats; calculated cosine curve of circadian variations is superimposed on the chronogram. Cosinor plot is shown at the botton. The circadian rhythm is represented by one vector originating at center of plot (pole). Length of vector represents rhythm’s amplitude. Vector direction indicates rhythm’s acrophase (highest point in rhythm). Circular region at tip of vector portrays joint confidence region (95%) for amplitude and acrophase. This region does not overlap the pole indicating a significant circadian rhythm. Arc described by tangents to region depicts conservative confidence interval for acrophase.
A nalitical procedures Determination of total glutathione content of liver prepared in 5q0 (w’v) thrichloroacetic acid in 0.01 N HCL was carried out as described by TIETZE (1969) with the modification of (1980). Hepatic cytOsO1ic glutathione stransferase activity was measured spectrophotometrically with 1-chloro-2, 4-dinitrobenzoic acid as substrate according to the methods of HABIG et al. (1974). Liver protein concentration was measured by the method Of et (1951) with bovine serum albumin as standard.
Statistical analysis All data were analysed for time-effects by the two ways analysis of variance (ANOVA) and for circadian
rhythm by a computerized inferential statistical et al., 1979),inmethod, the “single cosinor” (NELSON volving the fit of a 24 h cosine curve to averaged data series by the method of least squares. This method objectively determines the following information : - A probability or P value, which indicates the significance of the fit of the cosine curve to the data. If the P value was 0.05 or less, the fluctuation of the variable studied was presumed to be sinusoidal and not at random. - Three rhythmic parameters and their dispersions : these were designated as the acrophase, being either the time or angle of the crest of the fitted cosine curve; mesor being the cosinor determined overall 24-h mean, and amplitude being the half of the total cosine excursion. Both amplitude and mesor were expressed in the original units of the variable analyzed. In
GLUTATHIONE AND
4.5
GST : CIRCADIAN
85
RHYTHMS
-c
z c r =
4.0
0
E
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
03
3
-
S+ ko
3
N=6
3.5
-
3.0 i-I
I
I
I
I
I
L
I
I
I
FIG.2 . Circadian rhythm of liver glutathione. Chronogram (top) and cosinor test (botton) of liver glutathione concentration measured in six rats per time point. (For explanation see the legend of Figure 1).
addition to the mesor, amplitude, and acrophase, the coeficients of variation characterizing the data were calculated. The phasing was expressed in hours calculated in reference to local midnight. Results Although all animals exhibit a similar body weight, the hepatosomatic index [(liver wt/body wt) x 1001 shows a significant diurnal variation. The mean values at six different time points were :2.69, 2.75,2.92,2.87, 2.99, and 3.00%. Maximal hepatosomatic relations occurs during the dark period. The daily rhythm of cytosolic liver protein, shown in Figure 1 is typical of most rodents kept under the light-dark cycle indicated. The protein concentrations started to decrease at noon, reaching the lowest level in the midnight. The single cosinor analysis reveals the existence of a statistically significant circadian rhythm
characterized by a mesor of 94.96 mg/g liver (95% CI between 90.34 and 99.59), an amplitude of 16.30 mg/g liver (95% CI between 6.49 and 19.57) and an acrophase at 1310 h (95% CI between 1012 and 1557). CV = 93.04%. The diurnal rhythm of liver glutathione concentration, expressed as pmol/g liver, is shown in Figure 2. The minimum occurs in the dark period, gradually increasing as the light period begins. The mesor value is 3.75 pmol/g liver (95% CI between 3.63 and 3.87). The amplitude is 0.47 pmol/g liver and the acrophase occurs at 10.50 h (95% CI between 0848 and 1235). CV = 96.24%. Two activities of GST (nmol of conjugated CDNB/min. g liver and nmol of conjugated CDNB/min. mg protein) over a 24-h period are shown in Figure 3A and 3B. The diurnal patterns for both activities were quite similar. The two activities decreased during the dark period reaching the lowest values
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
86
M. J. TUSION, P. G O N Z ~ E Z ,P . LOPEZ, G. M. SALIDO AND J. A. MADRID
240
200 L
160
3-
Y
120
I,
a
I
I
12
I
I
16
I
I
20
0
4
a
TIME lFi\ FIG.3 . Diurnal changes in glutathione S-transferase activity. A : expressedper g of liver. B : expressedper mg of protein. Each point represents the mean i SEM of six animals. Continuous fine represents the best fitting cosine curve.
between OOOO and 0400 h, and then quickly increased to the high levels of activity observed in the light period. In both cases, non significant circadian rhythm can be detected by cosinor method because the data of enzyme activities are non-sinusoidal; however, the differences observed between data collected at different hours can be attributed to non-sinusoidal circadian rhythms, since revealed statistical differences bethe two-ways ANOVA tween time points. The GST activity shows an acrophase at 1325 h (nmol CDNB/min. g liver, Fig. 3A)and at 1312 h (nmol CDNB/min. mg protein, Fig. 3B) that partially coincides with those obtained for glutathione concentration. Discussion
Data by different authors indicate that activity of some hepatic enzymes and liver concentration of difet al., 1973;NOVELLO et al., ferent molecules (HOPKINS 1969) as well as the excretion of different biliary conet al., 1990)are stituents (DUANEet al., 1979;NAKANO modulated by food ingestion and follows a circadian pattern in the rat. With respect to glutathione metabolism it has been indicated that glutathione
concentration of liver (FAROOQUI & AHMED,1984; LESZCYSZYNSKA-BISSWANGER & PFAFF, 1985;SCHNELL et al., 1984), stomach (BODY et al., 1979), kidney & AHMED, 1984) and heart (DOROSHOW, (FAROOQUI 1979)exhibits a statistically significant diurnal variation in different species. Most of these time-dependent studies have been carried out with animals having spontaneous ad libitum or experimentallymodified feed patterns. However, periodical feeding can influence directly ("masking effect") the level of liver enzyme activities or indirectly, acting as a synchronizingagent, by means of a modification of circadian rhythm parameters, such as acrophase. In our experimental conditions we minimize the "masking" effects of food because every animal was fasted exactly 24 h prior to sacrifice. In this situation, the hepatic protein content shows a circadian rhythm which coincides with data previously published (SOLER et al., 1988). This protein rhythm is oppossed to hepatosomatic index oscillation and probably could be a reflex of the important rhythm & KHANDELWAL, 1985). in glycogen content (ROESLER Glutathione concentration also shows a diurnal variation coinciding with that of protein. The maintenance of hepatocellular glutathione is a dynamic
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by McMaster University on 01/07/15 For personal use only.
GLUTATHIONE AND
GST : CIRCADIAN RHYTHMS
process with a steady-state cellular concentration achieved by a balance between the rate of synthesis, catalized by glutamyl-cysteine synthetase and glutathione synthetase, the rate of utilization through redox and alkylating reactions and the rate of glutathione export from hepatocytes (NAKANO, 1990). The first enzymatic step is rate limiting in the synthesis of glutathione and largely limited by the availability of the free cysteine pool (VIRA et al., 1978): The degradation of glutathione is mainly accounted for sinusoidal efflux and in a minor part by consumption through conjugation reactions or enzymatic breakdown et al., 1985). (KAPLOWITZ GST activity also shows statistically significant diurnal variations, with a minimum during the maximal activity period of nocturnal rodents. This enzyme is involved in the detoxification of a large number of xenobiotics, including hepatic toxins such as carcinogens, a number of endogenous compounds including prostaglandins, leukotrienes, organic hydroperoxides and steroids are substrates for the GST (BOYER,1989). Thus, this rhythm can play an important role in the pharmacokinetic, toxicity and therapeutic effects of a number of drugs. Also the coincidence between diurnal changes in glutathione and in GST suggest that this enzymatic pathway can contribute to the circadian rhythm in glutathione concentration. However the possibility of changes in cysteine availability or glutathione export change remains open and further experiments are required to evaluate its contribution to circadian rhythm in the hepatic concentration of glutathione. Acknowledgments. - The authors are indebted to Mr. DENHAM WISDOMfor the advice on the English language.
References BECK,L. V., RIECK,V. D. & DUNCAN,B. (1958) Proc. SOC. Exp. Biol. 97, 229-231. BHATTACHARVA, R. D. (1981) in : Biological rhythms in structure and function, in : COSTO, P. M., VONMAYERSBACH, H., ScmvING, L. E. & PAULY, J. E. New York. BODY,S. C., SMAME,H. A. & BODY,M. R. (1979) Science 205, 1010-1012. BOYER,T. D. (1989) Hepatology 9, 486-496.
87 DIAZ-MUROZ,M., HERNANDEZ-MUAOZ, R., SUAREZ,J. & CHACOYA DE SANCHEZ,V. (1985) Neuroscience 16, 859-863. DOROSHOW, J. H., LOCKER,G. Y., BALDINGER, J. & MYERS,C. E. (1979) Res. Commun.Chem. Pathol. Pharmacol. 26,285-295. DUANE,W. C., GJLLVERSTADT, M. L. & WIEGAND,D. M. (1979) Am. J. Physiol. 236, R175-RI76. FAROOQUI, M. Y. & AHMED, A. E. (1984) Life Sci. 34, 2413-2418. GRLFFITH, 0 . W. (1980) Analyt. Biochem. 106, 207-212. HABIG,W. H., PALIST, M. J. & JAKOBY,W. B. (1974) J. Biol. Chem. 249, 7130-7139. HARDELAND, R., HOHMA", D. & RENSWG,L. (1973) J. Interdiscipl. Cycle Res. 4, 89-118. HOPKINS,H. A., BONNEY,R. J., WALKER,P. R., YAGER,J. D. & POTTER, V. R. (1973) Advanc. Enzyme Regul. 11, 169-191. ISAACS,J. & BINKLEY,F. (1977) Biochim. Biophys. Acta497, 192-204. KAp~owrrz,N., AW, T. Y., OOKHTENS,M. (1985) Ann. Rev. Pharmacol. Toxicol. 25, 715-744. KETTERER,B. (1986) Xenobiotic 10, 957-973. LESZCZYNSKA-BISSWANGER, A. & PEAEF, E. (1985) Biochem. Pharmacol. 34, 1635-1638. LOWRY,0 . H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-272. MEISTER,A. & ANDERSON, M. E. (1983) Ann. Rev. Biochem. 92, 711-760. NAKANO,A., TIETZ, P. S. & LARUSSON,N. F. (1990) Am. J. Physiol. 258, G653-G659. NELSON,W., TONG,L. Y. & HALBERG, F. (1979) Chronobiologia6, 305-323. NORTH,C., FEUERS, R. J., SCHEVING, L. E., PAULY,J. E., TSN, T. H. & CMCIANO,D. A. (1981) Am. J. Anat. 162, 183-199. NOVELLO, F., GUMAA, J. A. & MCLEAN,P. (1%9) Biochem. J. 111, 713-725. PREININGER, V., STEPAN,V., SERCE,J., KOJECKI,Z. & CERNOCH, M. (1961) Physiol. Bohemosl. 20, 307-311. R. L. (1985) Int. J. Biochem. 17, ROESLER, W. J. & KHANDELWAL, 81-85. SCHNELL, R. C., BOZIGIAN,H. P., DAVIES, M. H., MERRICK,B. A., PARK,K. S. & MCMJLLAN,D. A. (1984) Pharmacology 29, 149-157. SOLER,G., BAUTISTA,J. M., MADRID,J. A. & Sumo, G. M. (1988) Chronobiologia 15, 205-212. TIETZE,R. (1969) Analyt. Biochem. 27, 505-555. VANNOORDEN, C. J. F., BHATIACHARYA, R. D., VOOELS,I. M. C. & FRONIK, G. (1984) Chronobiologia 11, 131-138. H. & KREBS, H. A. (1978) Biochem. J. 170, VIAA,J., REGINALD, 627-630. J. A. MADRID, Department of Physiology and Pharmacology Faculty of Biology, University of Murcia Campus Espinardo, E-30100 Murcia. Spain.
