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Circadian rhythm of nuclear DNA in adult rat liver
Copyright 0 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved
Experimental Cell Research 76 (1973) 136142
CIRCADIAN
RHYTHM
OF NUCLEAR
DNA IN ADULT
RAT LIVER
J. R. RUBY,’ L. E. SCHEVING,2 S. B. GRAY’ and K. WHITE’ Department
of Anatomy, Wniversity
‘Louisiana State University Medical of Arkansas. Medical Center, Little
Center, New Orleans, La, 70119, and Rock, Ark. 72001, USA
SUMMARY Nuclei were isolated from adult rat livers at 2 h intervals over a 24 h period. The average DNA content per nucleus was determined in each nuclear suspension, It was found that there is a significant variation in the DNA content. The high point occurred during the late afternoon while the minimum was noted during the early morning. There also is a sharp peak of incorporation of 3H-thymidine between 0200 and 0400 hours. It is assumed that much of this incorporation cannot be counted for by mitosis since the mitotic index was found to be verv low (0.03-0.05 %). Possible mechanisms which could account for these observed cyclic variatfons are discussed.~’
A circadian rhythm in DNA synthesis in the livers of young, growing animals [2, 6, 141 and in regenerating livers of adult rodents [ 1, 2, 31 has been reported. One group of investigators [7, 14, 15, 171 has reported a circadian variation in the DNA concentration of the liver and in the UV absorbance measured by microspectrophotometric techniques in nuclei of hepatic cells in normal adult rats. The finding of a variation in DNA concentration in adult liver has not been widely accepted because it is commonly believed that the DNA content of adult liver is stable and any suggestions to the contrary frequently is thought to be influenced by technical factors. More recently, however, Echave-Llanos [6] using similar methodology has offered additional evidences to support this claim. The present investigation was an attempt to overcome certain technical objections by using isolated nuclei rather than pieces of whole liver to evaluate DNA concentration. The data presented here previously were reported in abstract form [21]. Exptl
Cell Res 76 (1973)
MATERIALS
AND METHODS
Animals All animals used were adult male Sprague-Dawley rats (weight range for each group is discussed in Results and Discussion section) which had been maintained for at least 7 days prior to each experiment in a carefully controlled environment. This included 2 animals per cage kept in a light-tight and sound deadened room maintained at 23” + 1°C. The room was illuminated with fluorescent light from 0600 to 1800 (CST) and darkened completely from 1800 to 0600. Rockland rat chow and tap water were available ad libitum. To minimize disturbances in the animal quarters, cage cleaning and replenishing of food and water were undertaken at 1400 on Monday, Wednesday and Friday. Because the four different experiments were conducted during June, July, November and March, we designated each experiment by the month in which it was performed. In the June and July experiments, 8 animals per time point were used while in the November and March experiments 8 and 10 respectively, were used per time point.
Isolation of nuclei At intervals of 2 h throughout each sampling day animals were taken from the standardized animal quarters to the autopsy room where they were rapidly decapitated and livers were obtained. Since the experimental protocol was restrictive in allowing only a limited amount of time to isolate the nuclei, a rapid, simple method using dilute solutions of citric acid was employed. The rats were killed by rapid decapitation and the livers removed and weighed. The livers
Circadian rhythm of DNA were homogenized in 0.05 M citric acid and spun at 1 500 g in a refrigerated International PR-2 centrifuge. The sediment was resuspended in 0.015 M citric acid and filtered through gauze. After centrifugation, the precipitate was resuspended and filtered through a double layer of flanelette. The nuclei were washed three times by centrifugation at 300 g and stored in the cold. Before chemical analysis, each suspension was washed two additional times.
Biochemical analysis Prior to chemical analysis, aliquots of nuclear suspensions were counted with a Coulter electronic particle counter according to the method described by Santen 1221.Multiple counts could be made with an error of approx. 1%. DNA content was determined by the diphenylamine technique of Burton [5], using a commerically prepared calf thymus DNA as a standard. All determinations were done in duplicate.
Radioactivity measurements In the November and March experiments, 7 of the 8 and 5 of the 10 animals used respectively at each time point were injected intraperitoneally with 3Hmethyl-thymidine (0.6 pCi/g of body weight; spec. act. was 19.7 Ci/@mole). The injected and noninjected animals were sacrificed 1 h after injection. At a later time, the isolated nuclei were washed twice in ice-cold 0.5 N perchloric acid (acid-soluble fraction) and the DNA subsequently extracted in hot perchloric acid. Aliquots were removed and measured for radioactivity in a Packard TriCarb liquid scintillation counter. Other aliquots were used for DNA determinations. For autoradiography, slides containing sections of liver were dipped in Kodak NTB 2 liquid emulsion and stored for 3 weeks to 6 months. The slides were processed in D-19 developer and hypo and stained with hematoxylin and eosin.
Microscopy For light microscopy, pieces of liver were removed from an identical area of each liver and fixed in 10 % buffered formalin. After dehydration the tissue was embedded in paraffin. Slides were processed directly in hematoxylin and eosin or processed for autoradiography. Mitotic counts were done by examining a minimum of 50 oil-immersion fields per liver. For electron microscopy, ten nuclear suspensions were selected at random and aliquots were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) followed by post-fixation in osmium tetroxide. The nuclear pellets were embedded in Maraglas and thin sectioned. The sections were stained with lead citrate and uranyl acetate and examined with a Philips 300 electron microscope.
Statistics In addition to analysing data by conventive student’s t-test, all data were analysed further by an inferential
131
statistical method designed to estimate ob.jectively a number of rhythmic parameters [lo]. This-was done by fitting a 24-hour cosine curve to the data by the method of least squares to obtain the following parameters: (1) Level (C,) the rhythm-adjusted computerdetermined overall 24 h mean value (equal to the arithmetic mean only if the data are equidistant); (2) amplitude (C) the distance from the level to the peak or trough of the cosine curve expressed in the same units as the variable analysed; (3) acrophase (0) the time when the data values are highest, hence the peak of the cosine curve expressed in degrees frommidnight where 1” = 4 min; 1s” = 1 h; 360” =24 h. A P value of 0.05 or less indicates that a data series is not fluctuating by chance alone. For a more detailed description of this method, the interested reader is referred to two papers by Halberg et al. [lo, II].
RESULTS AND DISCUSSION Fig. 1 is an electron micrograph of nuclei used in this study and attests to purity of the preparations. Fig. 2 demonstrates the normal variation in the rat liver weight over the 24-h span for the June and July experiments. The acrophases occurred at 08.48 (- 132”) and 08.36 (- 129’), respectively. Similar curves were seen for the November and March experiments, their acrophases occurred at 07.08 (- 107”) and 08.16 (- 124”), respectively. The overall mean acrophase for the four studies occurred at 08.12 (- 123”). These data demonstrate the reproducibility of the phasing of the liver weight rhythm from one study to another when animals are similarly standardized. Circadian variation in the rat liver is in the range of 10%. We have also found that mild stimuli, such as careless handling, prior to sacrifice can reduce the weight of the liver by another 10 y’ within 15 min subsequent to the application of the stimulus (unpublished results). Thus, it is possible with just careless handling to have a fluctuation of 20 % in liver weight. Such variation does question the reliability of analyses of rat liver components which are based on the amount per g or mg of liver wet weight. It must also be noted that the body weight of Exptl Cell Res 76 (1973)
138 J. R. Ruby et al.
Fig. I. An electron micrograph demonstrating the purity of a nuclear preparation used in this study. x 3 000.
Fig. 2. Abscissa: time of day (CST); ordinate: liver weight (g). Twenty-four hour cosine curve analysis is given below (see Materials and Methods). O”, local midnight, 15”, 1 h; 4”, 1 min.
Condition of experiment
Lx-&I,-06
P
Noise-tosignal (C2S.E.)
Level, Co (C&SE.)
Amplitude, C (C+S.E.)
Acrophase (0.95 confidence arc)
0.02
0.2S6
10.67+-0.18
0.90 kO.26
-132 (-100,
-164)
0.001
0.15*
10.74k 0.08
0.71 i-o.1 1
-129 (-111,
-147)
June Lo~-&w,~ July
Acrophase = Local midnight. Exptl Cell Res 76 (1973)
Circadian rhythm of DNA
time of day (CST); ordinate: DNA/nucleus. Fluctuation of DNA during 24 h cycle. Fig. 3. Abscissa:
Condition of experiment
P
Noise-tosignal @E./C)
La-xGs-os
0.02
0.28,
Nov. 1969 L-Jhww March 1970
0.01
0.16,
Level, C, (C+S.E.)
139
pg
Amplitude, C (Ct-S.E.)
Acrophase (0.95 confidence arc)
9.09 +0.08
0.40+0.11
- 274 ( - 242, - 305)
10.45+0.11
0.9010.15
- 288 (- 268, - 308)
Acrophase = Local midnight.
the animals was different from study to study. The June and July rats averaged 250 g whereas the November and March groups were older and averaged 400 and 550 g, respectively. Since it was the purpose of this study to determine whether a DNA rhythm occurred in older animals, where it is commonly accepted that there is virtually no mitotic activity, detailed studies of DNA synthesis were performed on the two older groups. The results of the analyses demonstrating the fluctuation in the average amount of DNA per nucleus in suspension for the November and March experiments are recorded in fig. 3. The acrophases of both studies occurred at similar times, 18.16 ( - 274”) and 19.12 ( -288”), respectively. The difference between the lowest and highest
recorded means in both studies was statistically significant. The 24 h mean DNA content of the March experiment was 1.36 pg higher than the experiment done in November. It is assumed that this increase is due to the larger and older animals used in March. The average content per nucleus in both instances, however, remains within the range of values reported by others [8, 221. The specific activity of DNA was determined in those animals which had received an injection of labelled thymidine. No significant activity was detected in the acid-soluble fraction. Since citric acid was used as the medium for isolation of the nuclei, the acidsoluble nucleotides were probably lost during preparation. Thymidine incorporation into DNA was Exptl Cell Res 76 (1973)
140 J. R. Ruby et al.
25 23
i ’
Fig. 4. Abscissa: time of day (CST); ordinate: cpm/mg
DNA x 1O-3. “H-thymidine cycle.
Condition of experiment
Lw--1&--0~
Nov. 1969 L-&,8--0e March 1970
incorporation
in DNA
over 24 h
P
Noise-Tosignal (SE/C)
Level, C, (C&S.E.)
Amplitude, C (C&SE.)
Acrophase (0.95 confidence arc)
0.23
0.54,
21.53 i0.92
2.43i1.31
-84
0.05
0.349
20.17+1.12
4.62k1.61
-71 (-34,
-109)
Acrophase = Local midnight.
noted at all points during the 24 h cycle with a significant increase between 02.00 and 05.00 hours, as indicated in fig. 4. The precise acrophases for the November and March experiments occurred at 05.36 ( - 84”) and 04.44 ( - 71”), respectively. The inverse relationship between DNA content and thymidine incorporation is shown in fig. 5. The sharp peak of incorporation has been reported by others in adult livers [7], in the livers of young immature rodents [2, 6, 91 and following partial hepatectomy [2, 3, 161. In the case of young growing livers and in regenerating livers, most of the incorporation can be attributed to replication of DNA prior to mitosis. However, it is commonly accepted that virtually no mitotic activity occurs in the adult liver 1131. During this study, all livers in the NovemExptl Cell Res 76 (1973)
ber and March experiments were examined for evidence of mitosis and labelled nuclei. An occasional mitotic figure and several completely labelled parenchymal cell nuclei were observed (fig. 6). The maximum mitotic index which was 0.05 % in November and 0.03 %, in March, occurred between 06.00 and 08.00 hours. All labelled cells were found in samples around 02.00 hours which corresponds to the time of peak incorporation of thymidine. It can be concluded, therefore, that the few cells which divide in adult liver do so by following the normal circadian pattern characteristic of young immature rodents. Such findings are in accord with a recent report by Rosene & Halberg on adult mouse liver
ra. Because we do see an occasional fully labeled parenchymal cell nucleus followed 4-6
Circadian rhythm of DNA
28
24 22
fkLYP
I2 0600
Fig. 5. Abscissa: time of day (CST); ordinate:
pg DNA/nucleus; (right) cpm/mg DNA x 1O-3.
(left)
Inverse relationship of DNA content/nucleus and specific activity.
hours later by the appearance of several mitotic figures, it must be assumed that some of the activity indicates DNA replication prior to mitosis although it is doubtful that the few nuclei undergoing mitosis can account fully for the high level of activity. One
141
could postulate, however, that the activity is the result of a normally occurring DNA repair replication similar to that observed in cells following UV irradiation [23]. One other possible mechanism which could produce the inverse relations between DNA incorporation content and 3H-thymidine would be an underlying cyclic variation in nuclease activity. A low nuclease activity during the light hours followed by increased activity during the dark period would cause the variation in DNA content which we have reported. Therefore, the peak incorporation of thymidine during the dark period would be suggestive of DNA turnover. In this regard, these results would tend to support the hypothesis of a ‘metabolic’ DNA [18, 191. The turnover-time of DNA in normal liver has been estimated to be 20-30 days at a rate of 3-5 % per day [18]. Another mechanism which could influence the average DNA content per nucleus is ‘ploidy shift’ over the 24-h period [4] although the extensive histological studies done during this investigation
Fig. 6. Autoradiograph showing two liver parenchymal cell nuclei. Nucleus on left is labelled with many grains demonstrating incorporation of 3H-thymidine. Exptl Cell Res 76 (1973)
142 J. R. Ruby et al. have not supported such an occurrence. In addition, it is possible that there are cyclic variations in nuclear fragility, a factor which has not been fully explored in this investigation. In this case the results would be a reflection of non-random sampling of nuclei due to breakage. This would effect the average content of DNA per nucleus especially since the liver contains a large percentage of polyploid nuclei. The authors wish to acknowledge the skillful assistante of Mr Tien-Hu Tsai. The work was supported by an NIH grant AM 15168 awarded to LES.
REFERENCES Bade, E G, Sadnik, I L, Pilgrim, C & Maurer, W, Exptl cell res 44 (1966) 676. l7$biroli, B & Potter, V R, Science 172 (1971)
Barium, C P, Jardetzky, C D & Halberg, F, Texas rep biol med 151 (1957) 139. Bucher, 0 & Suppan, P, The cellular aspects of biorhythms (ed H v Mayersbach) p. 126. Springer, New York (1967). 5. Burton, K, Biochem j 62 (1956) 315. 6. Echave-Llanos, J, devaccaro, M E E & Surur, J M, J interdiscipl cycle res 1 (1970) 161.
Exptl Cd Res 76 (1973)
7. Eling, W, The cellular aspects of biorhythms (ed H v Mayersbach) p. 105. Springer, New York (1967). 8. Falzone, J A, Barrows, C H & Yiengst, M J, Exptl cell res 26 (1962) 552. 9. Halberg, F, Barnum C P, Silber, R H & Bittner, J, Proc sot exptl biol med 97 (1958) 897. IO. Halberg, F, Tong, Y L & Johnson, E A, The cellular aspects of biorhythms (ed H v Mayersbath) p. 20. Springer, New York (1967). 11. Halberg, F, Johnson, E A, Nelson, W, Runge, W & Sothern, R, Physiol teacher 1 (1972) 1. 12. Horvath, G, Nature 200 (1963) 261. 13. Jackson, B, Anat ret 134 (1959) 365. 14. Jardetzky, C D, Barnum, C P & Halberg, R, Am j physiol 187 (1956) 3. 15. Jerusalem, C, The cellular aspects of biorhythms (ed H v Mayersbach) p. 115. Springer, New York (1967). 16. Looney, W B, Chang, L 0 and Banghart, F W, Proc natl acad sci US 57 (1967) 972. 17. Maversbach. H v. The cellular asuects of biorhythms, p. 87. Springer, New York-(1967). 18. Pelt, S R, Exptl geront 1 (1965) 215. 19. Roels, H, Int rev cytol 19 (1966) 1. 20. Rosene, G & Halberg, F, Bull all India inst med sci 4 (1970) 77. 21. Ruby, J R, Scheving, L E, Gray, S B & White, K, Proc IX inter tong anat, Leningrad, p. 152 (1970). 22. Santen, R J, Exptl cell res 40 (1965) 413. 23. Stockdale, F E & O’Neill, M C, J cell biol 52 (1972) 589. Received February 9, 1972 Revised version received May 19, 1972
Experimental Cell Research 76 (1973) 136142
CIRCADIAN
RHYTHM
OF NUCLEAR
DNA IN ADULT
RAT LIVER
J. R. RUBY,’ L. E. SCHEVING,2 S. B. GRAY’ and K. WHITE’ Department
of Anatomy, Wniversity
‘Louisiana State University Medical of Arkansas. Medical Center, Little
Center, New Orleans, La, 70119, and Rock, Ark. 72001, USA
SUMMARY Nuclei were isolated from adult rat livers at 2 h intervals over a 24 h period. The average DNA content per nucleus was determined in each nuclear suspension, It was found that there is a significant variation in the DNA content. The high point occurred during the late afternoon while the minimum was noted during the early morning. There also is a sharp peak of incorporation of 3H-thymidine between 0200 and 0400 hours. It is assumed that much of this incorporation cannot be counted for by mitosis since the mitotic index was found to be verv low (0.03-0.05 %). Possible mechanisms which could account for these observed cyclic variatfons are discussed.~’
A circadian rhythm in DNA synthesis in the livers of young, growing animals [2, 6, 141 and in regenerating livers of adult rodents [ 1, 2, 31 has been reported. One group of investigators [7, 14, 15, 171 has reported a circadian variation in the DNA concentration of the liver and in the UV absorbance measured by microspectrophotometric techniques in nuclei of hepatic cells in normal adult rats. The finding of a variation in DNA concentration in adult liver has not been widely accepted because it is commonly believed that the DNA content of adult liver is stable and any suggestions to the contrary frequently is thought to be influenced by technical factors. More recently, however, Echave-Llanos [6] using similar methodology has offered additional evidences to support this claim. The present investigation was an attempt to overcome certain technical objections by using isolated nuclei rather than pieces of whole liver to evaluate DNA concentration. The data presented here previously were reported in abstract form [21]. Exptl
Cell Res 76 (1973)
MATERIALS
AND METHODS
Animals All animals used were adult male Sprague-Dawley rats (weight range for each group is discussed in Results and Discussion section) which had been maintained for at least 7 days prior to each experiment in a carefully controlled environment. This included 2 animals per cage kept in a light-tight and sound deadened room maintained at 23” + 1°C. The room was illuminated with fluorescent light from 0600 to 1800 (CST) and darkened completely from 1800 to 0600. Rockland rat chow and tap water were available ad libitum. To minimize disturbances in the animal quarters, cage cleaning and replenishing of food and water were undertaken at 1400 on Monday, Wednesday and Friday. Because the four different experiments were conducted during June, July, November and March, we designated each experiment by the month in which it was performed. In the June and July experiments, 8 animals per time point were used while in the November and March experiments 8 and 10 respectively, were used per time point.
Isolation of nuclei At intervals of 2 h throughout each sampling day animals were taken from the standardized animal quarters to the autopsy room where they were rapidly decapitated and livers were obtained. Since the experimental protocol was restrictive in allowing only a limited amount of time to isolate the nuclei, a rapid, simple method using dilute solutions of citric acid was employed. The rats were killed by rapid decapitation and the livers removed and weighed. The livers
Circadian rhythm of DNA were homogenized in 0.05 M citric acid and spun at 1 500 g in a refrigerated International PR-2 centrifuge. The sediment was resuspended in 0.015 M citric acid and filtered through gauze. After centrifugation, the precipitate was resuspended and filtered through a double layer of flanelette. The nuclei were washed three times by centrifugation at 300 g and stored in the cold. Before chemical analysis, each suspension was washed two additional times.
Biochemical analysis Prior to chemical analysis, aliquots of nuclear suspensions were counted with a Coulter electronic particle counter according to the method described by Santen 1221.Multiple counts could be made with an error of approx. 1%. DNA content was determined by the diphenylamine technique of Burton [5], using a commerically prepared calf thymus DNA as a standard. All determinations were done in duplicate.
Radioactivity measurements In the November and March experiments, 7 of the 8 and 5 of the 10 animals used respectively at each time point were injected intraperitoneally with 3Hmethyl-thymidine (0.6 pCi/g of body weight; spec. act. was 19.7 Ci/@mole). The injected and noninjected animals were sacrificed 1 h after injection. At a later time, the isolated nuclei were washed twice in ice-cold 0.5 N perchloric acid (acid-soluble fraction) and the DNA subsequently extracted in hot perchloric acid. Aliquots were removed and measured for radioactivity in a Packard TriCarb liquid scintillation counter. Other aliquots were used for DNA determinations. For autoradiography, slides containing sections of liver were dipped in Kodak NTB 2 liquid emulsion and stored for 3 weeks to 6 months. The slides were processed in D-19 developer and hypo and stained with hematoxylin and eosin.
Microscopy For light microscopy, pieces of liver were removed from an identical area of each liver and fixed in 10 % buffered formalin. After dehydration the tissue was embedded in paraffin. Slides were processed directly in hematoxylin and eosin or processed for autoradiography. Mitotic counts were done by examining a minimum of 50 oil-immersion fields per liver. For electron microscopy, ten nuclear suspensions were selected at random and aliquots were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) followed by post-fixation in osmium tetroxide. The nuclear pellets were embedded in Maraglas and thin sectioned. The sections were stained with lead citrate and uranyl acetate and examined with a Philips 300 electron microscope.
Statistics In addition to analysing data by conventive student’s t-test, all data were analysed further by an inferential
131
statistical method designed to estimate ob.jectively a number of rhythmic parameters [lo]. This-was done by fitting a 24-hour cosine curve to the data by the method of least squares to obtain the following parameters: (1) Level (C,) the rhythm-adjusted computerdetermined overall 24 h mean value (equal to the arithmetic mean only if the data are equidistant); (2) amplitude (C) the distance from the level to the peak or trough of the cosine curve expressed in the same units as the variable analysed; (3) acrophase (0) the time when the data values are highest, hence the peak of the cosine curve expressed in degrees frommidnight where 1” = 4 min; 1s” = 1 h; 360” =24 h. A P value of 0.05 or less indicates that a data series is not fluctuating by chance alone. For a more detailed description of this method, the interested reader is referred to two papers by Halberg et al. [lo, II].
RESULTS AND DISCUSSION Fig. 1 is an electron micrograph of nuclei used in this study and attests to purity of the preparations. Fig. 2 demonstrates the normal variation in the rat liver weight over the 24-h span for the June and July experiments. The acrophases occurred at 08.48 (- 132”) and 08.36 (- 129’), respectively. Similar curves were seen for the November and March experiments, their acrophases occurred at 07.08 (- 107”) and 08.16 (- 124”), respectively. The overall mean acrophase for the four studies occurred at 08.12 (- 123”). These data demonstrate the reproducibility of the phasing of the liver weight rhythm from one study to another when animals are similarly standardized. Circadian variation in the rat liver is in the range of 10%. We have also found that mild stimuli, such as careless handling, prior to sacrifice can reduce the weight of the liver by another 10 y’ within 15 min subsequent to the application of the stimulus (unpublished results). Thus, it is possible with just careless handling to have a fluctuation of 20 % in liver weight. Such variation does question the reliability of analyses of rat liver components which are based on the amount per g or mg of liver wet weight. It must also be noted that the body weight of Exptl Cell Res 76 (1973)
138 J. R. Ruby et al.
Fig. I. An electron micrograph demonstrating the purity of a nuclear preparation used in this study. x 3 000.
Fig. 2. Abscissa: time of day (CST); ordinate: liver weight (g). Twenty-four hour cosine curve analysis is given below (see Materials and Methods). O”, local midnight, 15”, 1 h; 4”, 1 min.
Condition of experiment
Lx-&I,-06
P
Noise-tosignal (C2S.E.)
Level, Co (C&SE.)
Amplitude, C (C+S.E.)
Acrophase (0.95 confidence arc)
0.02
0.2S6
10.67+-0.18
0.90 kO.26
-132 (-100,
-164)
0.001
0.15*
10.74k 0.08
0.71 i-o.1 1
-129 (-111,
-147)
June Lo~-&w,~ July
Acrophase = Local midnight. Exptl Cell Res 76 (1973)
Circadian rhythm of DNA
time of day (CST); ordinate: DNA/nucleus. Fluctuation of DNA during 24 h cycle. Fig. 3. Abscissa:
Condition of experiment
P
Noise-tosignal @E./C)
La-xGs-os
0.02
0.28,
Nov. 1969 L-Jhww March 1970
0.01
0.16,
Level, C, (C+S.E.)
139
pg
Amplitude, C (Ct-S.E.)
Acrophase (0.95 confidence arc)
9.09 +0.08
0.40+0.11
- 274 ( - 242, - 305)
10.45+0.11
0.9010.15
- 288 (- 268, - 308)
Acrophase = Local midnight.
the animals was different from study to study. The June and July rats averaged 250 g whereas the November and March groups were older and averaged 400 and 550 g, respectively. Since it was the purpose of this study to determine whether a DNA rhythm occurred in older animals, where it is commonly accepted that there is virtually no mitotic activity, detailed studies of DNA synthesis were performed on the two older groups. The results of the analyses demonstrating the fluctuation in the average amount of DNA per nucleus in suspension for the November and March experiments are recorded in fig. 3. The acrophases of both studies occurred at similar times, 18.16 ( - 274”) and 19.12 ( -288”), respectively. The difference between the lowest and highest
recorded means in both studies was statistically significant. The 24 h mean DNA content of the March experiment was 1.36 pg higher than the experiment done in November. It is assumed that this increase is due to the larger and older animals used in March. The average content per nucleus in both instances, however, remains within the range of values reported by others [8, 221. The specific activity of DNA was determined in those animals which had received an injection of labelled thymidine. No significant activity was detected in the acid-soluble fraction. Since citric acid was used as the medium for isolation of the nuclei, the acidsoluble nucleotides were probably lost during preparation. Thymidine incorporation into DNA was Exptl Cell Res 76 (1973)
140 J. R. Ruby et al.
25 23
i ’
Fig. 4. Abscissa: time of day (CST); ordinate: cpm/mg
DNA x 1O-3. “H-thymidine cycle.
Condition of experiment
Lw--1&--0~
Nov. 1969 L-&,8--0e March 1970
incorporation
in DNA
over 24 h
P
Noise-Tosignal (SE/C)
Level, C, (C&S.E.)
Amplitude, C (C&SE.)
Acrophase (0.95 confidence arc)
0.23
0.54,
21.53 i0.92
2.43i1.31
-84
0.05
0.349
20.17+1.12
4.62k1.61
-71 (-34,
-109)
Acrophase = Local midnight.
noted at all points during the 24 h cycle with a significant increase between 02.00 and 05.00 hours, as indicated in fig. 4. The precise acrophases for the November and March experiments occurred at 05.36 ( - 84”) and 04.44 ( - 71”), respectively. The inverse relationship between DNA content and thymidine incorporation is shown in fig. 5. The sharp peak of incorporation has been reported by others in adult livers [7], in the livers of young immature rodents [2, 6, 91 and following partial hepatectomy [2, 3, 161. In the case of young growing livers and in regenerating livers, most of the incorporation can be attributed to replication of DNA prior to mitosis. However, it is commonly accepted that virtually no mitotic activity occurs in the adult liver 1131. During this study, all livers in the NovemExptl Cell Res 76 (1973)
ber and March experiments were examined for evidence of mitosis and labelled nuclei. An occasional mitotic figure and several completely labelled parenchymal cell nuclei were observed (fig. 6). The maximum mitotic index which was 0.05 % in November and 0.03 %, in March, occurred between 06.00 and 08.00 hours. All labelled cells were found in samples around 02.00 hours which corresponds to the time of peak incorporation of thymidine. It can be concluded, therefore, that the few cells which divide in adult liver do so by following the normal circadian pattern characteristic of young immature rodents. Such findings are in accord with a recent report by Rosene & Halberg on adult mouse liver
ra. Because we do see an occasional fully labeled parenchymal cell nucleus followed 4-6
Circadian rhythm of DNA
28
24 22
fkLYP
I2 0600
Fig. 5. Abscissa: time of day (CST); ordinate:
pg DNA/nucleus; (right) cpm/mg DNA x 1O-3.
(left)
Inverse relationship of DNA content/nucleus and specific activity.
hours later by the appearance of several mitotic figures, it must be assumed that some of the activity indicates DNA replication prior to mitosis although it is doubtful that the few nuclei undergoing mitosis can account fully for the high level of activity. One
141
could postulate, however, that the activity is the result of a normally occurring DNA repair replication similar to that observed in cells following UV irradiation [23]. One other possible mechanism which could produce the inverse relations between DNA incorporation content and 3H-thymidine would be an underlying cyclic variation in nuclease activity. A low nuclease activity during the light hours followed by increased activity during the dark period would cause the variation in DNA content which we have reported. Therefore, the peak incorporation of thymidine during the dark period would be suggestive of DNA turnover. In this regard, these results would tend to support the hypothesis of a ‘metabolic’ DNA [18, 191. The turnover-time of DNA in normal liver has been estimated to be 20-30 days at a rate of 3-5 % per day [18]. Another mechanism which could influence the average DNA content per nucleus is ‘ploidy shift’ over the 24-h period [4] although the extensive histological studies done during this investigation
Fig. 6. Autoradiograph showing two liver parenchymal cell nuclei. Nucleus on left is labelled with many grains demonstrating incorporation of 3H-thymidine. Exptl Cell Res 76 (1973)
142 J. R. Ruby et al. have not supported such an occurrence. In addition, it is possible that there are cyclic variations in nuclear fragility, a factor which has not been fully explored in this investigation. In this case the results would be a reflection of non-random sampling of nuclei due to breakage. This would effect the average content of DNA per nucleus especially since the liver contains a large percentage of polyploid nuclei. The authors wish to acknowledge the skillful assistante of Mr Tien-Hu Tsai. The work was supported by an NIH grant AM 15168 awarded to LES.
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