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Age-related changes in total dna and rna and incorporation of uridine and thymidine in rat liver, kidney and spleen
00?0.71Ix.x3 53.00+0.00 Copyright 0 IYX3 Per&tmon Press Ltd
I,II. J. B~oclr~,,~~. Vol. IS. No. 9. pp. I103 1107. 1983 Printed III GreatBritain. All rights reserved
AGE-RELATED CHANGES IN TOTAL DNA AND RNA AND INCORPORATION OF URIDINE AND THYMIDINE IN RAT LIVER, KIDNEY AND SPLEEN L~JIGI MESSINEO.’CHARLES W. DENKO’ and MARK PEITRICE&* ‘Cleveland State University. ‘Scott Research Laboratory,
** Department of Biology. Cleveland. OH 441 15, U.S.A. Fairview General Hospital, Cleveland. OH 441 Il. U.S.A.
Abstract-l. Total content of DNA and RNA in liver. kidnev and spleen were measured in young and aged rats. At the same time the incorporation of [“C]thymid~ne, a DNA precursor, and C3H]uridine. an RNA precursor. were also determined. 2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporntion of precursors. 3. Young animals have more DNA per organ relative to RNA. with kidney :tnd spleen DNA showing a decrease between ~l~~turit~ and senescence. 4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [“Hluridine uptake decreases. 5. Liver polyploid differentiation, and [“Clthymidine and [‘Hluridine uptake. are correlated. 6. In kidney, incorporation of [‘H]uridine is inversely related to [‘“Clthymidine incorporation,
INTRODUCTION is no basic line of reference on changes in total DNA and RNA. or in the incorporation of E3H]uridine and [‘4C]thymidine during development. maturity and aging in the liver. spleen and kidney of Sprague-Dawley rats. This study establishes this line of reference. Growth and development of an organism requires synthesis and destruction of DNA and RNA. This synthesis and destruction reaches an equilibrium at maturity. The equilibrium is altered during aging. Age related changes in the incorporation of j’4C]thymidine, a DNA precursor, and of [“HIuridine. an RNA precursor. are related to gene activity regardless of possible limiting causes. Thus. if aging is regarded as a breakdown of the steady state equilibrium, the determination of age related changes in different organs may provide ;I gauge for future aging There
modification
studies.
The liver was selected for study because it participates in much of metabolism, while the kidney removes metabolites. The spleen was chosen because of its role in immunity. MATERIALS
AND METHODS
4rtirmfi.~
Male Sprague-Dawley rats (Flow Labotxories. Dublin. Virginia) were maintained under controlled temperature t23’C) and fed commercial pelleted rodent food (Purina Lab Chow, St Louis. Missouri) and tap water ~rl tihirtmr. The rats were killed when they rexhed 20, 40, 90, 220. 350. 550 and 700 days of itge. At least IO mts were studied
* Author
to whom reprint
requests
should
be addressed.
individually for each age group, except for the 700-day group which contained five animals. Weight vs age profiles (Fig. 1) showed normal growth for this strain of rats.
Rats were injected intraperitoneally with 2 /tCi/lOOg of body weight of [“‘Clthymidine 24 hr before sacrifice and with 10 /tCiilOO g of [‘Hluridine (International Chemical and Nuclear Corp.. Irvine, California) 4 hr prior to sacrifice. This time schedule was chosen to minimize the tritium tag lability for [3H]ttridine, an isotope that is less stable than [‘“C’lthymidine tagged at the C-2 position.
After ctheri7~~tion, all blood and unreacted isotopes were removed by whole body perfusion with isotonic saline through cathcrization of the left ventricle and cutting of the right atria and vena cava. The liver. spleen and kidney were cleaned of adhering tissue and separately placed in isotonic saline solution.
The initial homogenization was performed in saline with a Potter Elvehjem wide clearance glass homogenizer and a Sears 3,‘X” variable speed drill (0-I 200 rev:‘min). The suspensions were filtered through cotton gauze, adjusted to a final coI~centrt~tion of lo”,, trichloroacetic acid (TCA) using cold 50”,, TCA. and centrifuged for 10min at 5OOOrev,‘min in a superspeed refrigerated (4’C) centrifuge (Sorvall. Newton. CN). The supernatant fluid was discarded and the pellet mixed with cold IO”,, TCA and centrifuged again. This was repeated twice more to remove unre&d ;sotopc. Cold TCA precipitates highly polymcrired DNA and RNA leaving oligonucleotides and free nucleotides in solution. On long standing. RNA has a tendency to go in solution in TCA. To avoid this possibilitv. the cxDosure to TCA was . kept to a minimum constant time.
f.‘igtire
I sho\+s
Sprague
the
Dawleq
growth
in grams
of rats
in the
used in these studies.
colony
Figure 2 Illustrates total li\cr DNA and RNA content in milligrams \s ago in cia~s. The amount of RNA cacccds that of DNA by about five to one. The ovcrnll trend 01‘total RNA 15 tu increase at all ages except for the X0-da> (70.3 mg SD 37.5) and 550-da\; (101.7 tnp SD 371 pcrlod. The ratlo of DNA ‘RNA drops from 20 to 40 days. it increases afterward peaking at 350 days and. strhs~quentl~. dccrcases until 700 days.
Figure .3 sho\+s counts per minute per milligrum (counts min per mg) of DNA and RNA. Thqmidino incorporation sho\\s an almost linear drop from 20 da>s (I 137 counts min per mg SD 397) tc> YO da!s (412 counts min per mg SD 103) (P < 0.05). ;I slowi decline from YO to 350 dabs 1I76 counts, min per mg SD 3) I P < 0.05; and ;I slight increase from 31) to 700 days (I 7S co~~nts min per mg SD 51 ). RNA [‘H]uriclinc 1175
Incorporation
cotints
iii111 pt‘r
increxcs
11-g SD
Xi)
from to
40
20
clays
clans
(331
SD 15.5) IP < 0.05;. dtxrcascs from 40 to YO da!s (I X2 counts min per mg SD 45). peaks at 220 Ja!s (17X counts min per mg SD 2YO) and shows ;I slou do\+nlvard trend frotn 220 days to 700 da!s (155 COLITIS. min per tng SD 96) ;P c 0.05;. The ratio of [“C’jth!midinc to [‘Hluridintz uptake start\ high aind decreases signilicantlv until 350 da~3 of :rgc. .Phc I-atio thcii increases until 700 dais. counts
min
Total versus
RI\;A
per
kidnc!
mg
DNA
age in days inuuws
and
RNA
content
IS illustrated
DNA,
l’aster than
in milligrams
in Figure 3. Kidnq though both peak at
I /-. I
I--_._,/’
,.’
--._
-..I
,I’
I’ I’
100
2°C
300
4oc
Age
500
600
(days)
700
800
9
Age changes
in rat liver. kidney
c
/200 Ii00
-
1000
-
900
-
800
-
0
Figure 5 illustrates the incorporation of radiolabeled [14C]thymidine and [‘Hluridine expressed as counts per minute per milligram of DNA and RNA. The incorporation of [liC]thymidine into DNA is highest at 20 days (726 counts,nlin per mg SD 173) and declines to its lowest level at 350 days (247 SD 46) ff < 0.05:. and from there increases until 550 days (488 counts~min per mg SD 248) :P < 0.05;. RNA C3H]uridine incorporation increases from 20 days (205 counts:min per mg SD X0) to 350 d;tys (714 counts,/min per mg SD I 13) [P < 0.05 ). decrelises from 350 days to 700 days (614 countsjmin per mg SD 90). The ratio of [‘“Clthymidinc to [“Hluridine uptake starts high and decreases to its lowest value at 350 days of age. The ratio then changes direction and increases untii 550 days and then decreases again slightly at 700 days.
1
100
.
200
*
.
.
400
300
1
500
Age
600
.
roe
0
*
800900
(days)
F-ig. 3. The plot of liver uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = [2-‘Y‘]thymidine; dashed line = [G3H]uridine; thin solid
line = ratio incorporation
[‘4Cjthymidine/[3H]uridine.
50..
0
1IO5
and spleen
, 100
Figure 6 illustrates total DNA and RNA content (mg) vs age in days of rat spleen. Both total DNA and RNA increase with age until 350 days (DNA 10.3 SD 1.2, RNA 12.8 SD 1.7). The DNA content reaches its highest level at 350 days and then declines until 700 days (6.8 mg SD 1) ;P < 0.05: while the level of RNA shows an increase throughout. In the spleen of 20 and 40 day oid rats the amount of DNA exceeds RNA. However, this situation reverses after 40 days. relative to that of Thymidine incorporation C3H]uridine (Fig. 7) is much higher in all age groups. The highest level of both [‘4C]thymidine and [“Hluridine incorporation is at 20 days (thymidine 2889 countsjmin per mg SD 898. uridine 927 counts: min per mg SD 425) then both values show it drop from 20 to 40 days (thymidine 671 SD 374 {P < O.OSi. uridine 426 SD 164 {P < 0.05)). The ratio
, 200
300
400 Ag8
500
600
700
800
!
idOySi
f-ig. 4. The plot of total kidney DNA and RNA content in milligrams (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = DNA; dashed line = RNA: thtn solid line = ratio DNA/RNA, 0
350 days (RNA 23.5 mg SD 3.3, DNA Il.3 mg SD 1.7). After 350 days. both kidney RNA and DNA shows a slow progressive decline to a lower level at 700 days (RNA 18.9 mg SD 2.5, DNA 8.2 mg SD 2.3). The ratio of DNA to RNA is highest at 20 days, and increases again at 220 days, to a level which remains constant until 700 days.
,
,
,
,
,
,
,
,
100
200
300
400
500
600
700
8009
Age
(days)
Fig. 5. The plot of kidney uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = [2-“%]thymidine; dashed line = [6-3H]uridine; thin solid line = ratio incorporation [‘JC]thymidine/[3H]uridine.
LIIIGI Mtsww~
a IO0
200
300
400 Age
500
600
700
800
(do&)
Fig. 6. The ploi of total spleen DNA and RNA content in miihgrams (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = DNA: dashed line = RNA: thin solid line = ratio DNA’RNA.
of [‘~C]thymid~ne~[3H]uridine shows that the drop in [3H]uridine relative to [“%Z]thymidine is larger (Fig. 7). DISCC’SSION It has heen shown that in the liver there is a relationship hetueen the age of an animal and the transformation of hepatocytes from u diploid to polyploid state. S\n.artz (1956) postulated that this transf~~rnl~lti~~llis an increasing function during the first 20 yr of life in humans. Van Bezooijen et td. (1972) found that in rat the production of the polyploid state in liver peaked at about one year. When one compares the life cxpcctancy of rats and humans ntf see that the polyploid state occurs during the equivalent ln~itur~~tion age. Correlation of the results of this study with the above data leads us to the hypothesis that the production of a polyploid state is possibly related to dramatic lowering of [‘“Clthymidine incorporation (Fig. 3) in the liver. In early life, there are potentially a greater number of liver cells able to undergo polyploidy. This means higher DNA synthesis and [‘?Jthymidinc incorporation. In older animals, there would be fewer liver cells able to undergo polyploidy. This would be rellectcd in the lowering of the [“%I’]thymidine incorporation rate. Another explanation for some of the initial lowering in [‘JC]thymidine may be the observation of (Greengard (‘t tri.. 1972) that production of perinatal hematopoetic tissue in liver stops soon after birth. This decrcasc coincides with the decrease in DNA: RNA ratio between 20- to 40-day-old rats (Fig. 2) and also with the decrease in [‘“C]thymidine [“Hluridine ratio during this same period (Fig. 3).
c’t trl.
It is of interest that the total quantity of liver DNA shows an increase while the rate of [‘~C]thyrn~dilie incorporation is fatling. This may mean that 21 control point early in life sets the developmental program of the liver. The [“Hluridine uptake. unlike that of [‘4C]thymidine, reaches a peak at 200 days and consequently decreases to 700 days (Fig. 3). Since the total RNA content shows an increase. the catabolic rate for RNA destruction may be decreasing. The kidney (Fig. 4) shows that total DNA relative to RNA is highest at 20 days. This reflects ;I high nuclear to cytoplasmic ratio. As an animal matures. total DNA doubles by 90 days, while total RNA quadruples. From 90 to 220 days. kidney DNA shows a lower rate of increase. while RNA declines. The drop in total RNA. from 90 days to 220 days, may be a rcsponse to sexual m~~tur~ltion and may reflect an increase in RNA catabolism since synthesis increases (Fig. 4). After 350 days the total content of DNA and RNA, in kidneys (Fig. 4) drops by about lo”,,. The decline of RNA and DNA during this time span was statistically significant j P < 0.05 j and follows the observed decrease in total kidney weight. In the human kidney, maximum size. 270 g, is reached by the third to fourth decade and declines to I85 g by ninth (Epstein. 1979). This loss is mainly cortical and is associated with renal vascular changes. sclerosis and a collapse of the glomerular tuft. The increase in DNA synthesis from 350 to 700 days may be a result of fibroblast proliferation due to autoimmune related damage or a compensatory response to glomerular loss. The spleen is composed of a heterogenous mixture of cells. The initial increase in total RNA content (Fig. 6) ma! correspond with attainment of immunocompe-
-e
* L____-----_
I
--_________---
_.-
I00 200 300 400 500 600 100 800 900° Age (days) Fig. 7. The plot of spleen uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in daqs. Solid line = [2-“Vlthymidine: dashed line = [6-“Hluridine: thin solid lint = ratio incorporation ~‘~C]thymidine,C’H]uridlnc.
Age
I107
changes in rat Ii\er. kidney and spleen
tence in the RNA-rich plasma cell line. The relative decrease in [“Hluridine incorporation (Fig. 6) implies a negative control point during the neonatal period that ultimately sets the level of total RNA at maturity (Fig. 6). At 90 days (Fig. 7). [‘4C]thymidine incorporation decreases. possibly due to sexual development. The decrease in total quantity of DNA (Fig. 6) after 350 days is statistically significant [P < 0.05), and may express a generalized decline in immunocompetence with age. The increased [“‘Clthymidine incorporation after 350 days may reflect a compensatory response to the drop in total DNA. Thymidine incorporation relative to that of [-‘Hluridine (Fig. 7) is much higher at all age groups. The highest level of both [‘4C]thymidine and [ ‘Hluridine incorporation is at 20 days. Both values show a drop from 20 to 40 days. The ratio of [“Clthymidine [“Hluridine shows that the drop in [ ‘Hluridine relative to [14C]thymidine is larger (Fig. 71. Total DNA and RNA ratio show an almost contenuous decrease (Fig. 6). These data support the view that the ratio of nucleus (DNA) to cytoplasm (RNA) decreases with differentiation and perhaps aging (Minot. 1907).
We would like to submit the following conclusions as a result of our studies. Total DNA increases up to 350 days of age in the liver. kidney and spleen, and the rate of incorporation ol’thymidine decreases from birth to 350 days of age. In the life span of rats. the period corresponding to 350 days of age seems to be a turning point where total DNA per organ declines; paradoxically, the rates of thymidine incorporation increases. This may be an attempt by the organism to re-establish the equilibrium of maturity.
Similarly, the total amount of RNA in liver, kidney and spleen increases from birth to 350 days of age. However, the pattern of uridine incorporation in each organ is different. In the liver. the rate of uridine incorporation increases up to 350 days of age and thereafter decreases. In the kidney, uridine incorporation increases up to 350 days of age and then remains constant. This ma] be the result of a compensatory incrcasc in activity related to the loss of glomeruli. In the spleen. the rate of uridine incorporation is constant after the first 40 days of life.
REFERENCES
Geigy. Scicwtific Ttrhlrs. 5th edn. p. 39. K~qer. New York (1959). EPSTEIN M. (1979) Etfects of aging on the kidney. F&Z Proc. 38. 168-171. GK~ENGARI) 0.. FEIXRMAE;M. & Knox W. F. (1972) Cvtomorphometry of developing rilt liver and Its npplica;ion to enzymic diflerentintion. J. w/l. Biol. 52. 761 272. MFSSINW L. (1972) Interferences in deoxyribose detcrmination by the cysteine-HCI sulphuric acid method /,lt. J. Biwhw. 3, 53 I 536. MESSI~*;FO L. & D’AMI(.o J. (1972) A test for ribose determination without interference from dcoxyribose. Irlr. J. BioclIr/?l. 3. 35 I-356. MINOTC. S. (1907) The problem of age, gro\%th and death fop. Sci. hfon. 71. June 481 496: August 97 120: December 509- 523. SW~RTZ F. J. (I 956) The development in the human hver of multlple desoxyrlbose nucleic acid (DNA) cI;lsses and their relationship to the age of the individual. C/I~I~IOSOU,~,8, 53-72. VAN BEZOOIJANC. F. A.. DELEEW-ISRAEL F. R. & HOLLAXER C. F. (1972) On the role of hepatic cell polyploidy in changes in liver function with age and following partial hepatectomy. Mrcl~. Acqeirq LIrr. 1. ?I 356. Docu~mwta
I,II. J. B~oclr~,,~~. Vol. IS. No. 9. pp. I103 1107. 1983 Printed III GreatBritain. All rights reserved
AGE-RELATED CHANGES IN TOTAL DNA AND RNA AND INCORPORATION OF URIDINE AND THYMIDINE IN RAT LIVER, KIDNEY AND SPLEEN L~JIGI MESSINEO.’CHARLES W. DENKO’ and MARK PEITRICE&* ‘Cleveland State University. ‘Scott Research Laboratory,
** Department of Biology. Cleveland. OH 441 15, U.S.A. Fairview General Hospital, Cleveland. OH 441 Il. U.S.A.
Abstract-l. Total content of DNA and RNA in liver. kidnev and spleen were measured in young and aged rats. At the same time the incorporation of [“C]thymid~ne, a DNA precursor, and C3H]uridine. an RNA precursor. were also determined. 2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporntion of precursors. 3. Young animals have more DNA per organ relative to RNA. with kidney :tnd spleen DNA showing a decrease between ~l~~turit~ and senescence. 4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [“Hluridine uptake decreases. 5. Liver polyploid differentiation, and [“Clthymidine and [‘Hluridine uptake. are correlated. 6. In kidney, incorporation of [‘H]uridine is inversely related to [‘“Clthymidine incorporation,
INTRODUCTION is no basic line of reference on changes in total DNA and RNA. or in the incorporation of E3H]uridine and [‘4C]thymidine during development. maturity and aging in the liver. spleen and kidney of Sprague-Dawley rats. This study establishes this line of reference. Growth and development of an organism requires synthesis and destruction of DNA and RNA. This synthesis and destruction reaches an equilibrium at maturity. The equilibrium is altered during aging. Age related changes in the incorporation of j’4C]thymidine, a DNA precursor, and of [“HIuridine. an RNA precursor. are related to gene activity regardless of possible limiting causes. Thus. if aging is regarded as a breakdown of the steady state equilibrium, the determination of age related changes in different organs may provide ;I gauge for future aging There
modification
studies.
The liver was selected for study because it participates in much of metabolism, while the kidney removes metabolites. The spleen was chosen because of its role in immunity. MATERIALS
AND METHODS
4rtirmfi.~
Male Sprague-Dawley rats (Flow Labotxories. Dublin. Virginia) were maintained under controlled temperature t23’C) and fed commercial pelleted rodent food (Purina Lab Chow, St Louis. Missouri) and tap water ~rl tihirtmr. The rats were killed when they rexhed 20, 40, 90, 220. 350. 550 and 700 days of itge. At least IO mts were studied
* Author
to whom reprint
requests
should
be addressed.
individually for each age group, except for the 700-day group which contained five animals. Weight vs age profiles (Fig. 1) showed normal growth for this strain of rats.
Rats were injected intraperitoneally with 2 /tCi/lOOg of body weight of [“‘Clthymidine 24 hr before sacrifice and with 10 /tCiilOO g of [‘Hluridine (International Chemical and Nuclear Corp.. Irvine, California) 4 hr prior to sacrifice. This time schedule was chosen to minimize the tritium tag lability for [3H]ttridine, an isotope that is less stable than [‘“C’lthymidine tagged at the C-2 position.
After ctheri7~~tion, all blood and unreacted isotopes were removed by whole body perfusion with isotonic saline through cathcrization of the left ventricle and cutting of the right atria and vena cava. The liver. spleen and kidney were cleaned of adhering tissue and separately placed in isotonic saline solution.
The initial homogenization was performed in saline with a Potter Elvehjem wide clearance glass homogenizer and a Sears 3,‘X” variable speed drill (0-I 200 rev:‘min). The suspensions were filtered through cotton gauze, adjusted to a final coI~centrt~tion of lo”,, trichloroacetic acid (TCA) using cold 50”,, TCA. and centrifuged for 10min at 5OOOrev,‘min in a superspeed refrigerated (4’C) centrifuge (Sorvall. Newton. CN). The supernatant fluid was discarded and the pellet mixed with cold IO”,, TCA and centrifuged again. This was repeated twice more to remove unre&d ;sotopc. Cold TCA precipitates highly polymcrired DNA and RNA leaving oligonucleotides and free nucleotides in solution. On long standing. RNA has a tendency to go in solution in TCA. To avoid this possibilitv. the cxDosure to TCA was . kept to a minimum constant time.
f.‘igtire
I sho\+s
Sprague
the
Dawleq
growth
in grams
of rats
in the
used in these studies.
colony
Figure 2 Illustrates total li\cr DNA and RNA content in milligrams \s ago in cia~s. The amount of RNA cacccds that of DNA by about five to one. The ovcrnll trend 01‘total RNA 15 tu increase at all ages except for the X0-da> (70.3 mg SD 37.5) and 550-da\; (101.7 tnp SD 371 pcrlod. The ratlo of DNA ‘RNA drops from 20 to 40 days. it increases afterward peaking at 350 days and. strhs~quentl~. dccrcases until 700 days.
Figure .3 sho\+s counts per minute per milligrum (counts min per mg) of DNA and RNA. Thqmidino incorporation sho\\s an almost linear drop from 20 da>s (I 137 counts min per mg SD 397) tc> YO da!s (412 counts min per mg SD 103) (P < 0.05). ;I slowi decline from YO to 350 dabs 1I76 counts, min per mg SD 3) I P < 0.05; and ;I slight increase from 31) to 700 days (I 7S co~~nts min per mg SD 51 ). RNA [‘H]uriclinc 1175
Incorporation
cotints
iii111 pt‘r
increxcs
11-g SD
Xi)
from to
40
20
clays
clans
(331
SD 15.5) IP < 0.05;. dtxrcascs from 40 to YO da!s (I X2 counts min per mg SD 45). peaks at 220 Ja!s (17X counts min per mg SD 2YO) and shows ;I slou do\+nlvard trend frotn 220 days to 700 da!s (155 COLITIS. min per tng SD 96) ;P c 0.05;. The ratio of [“C’jth!midinc to [‘Hluridintz uptake start\ high aind decreases signilicantlv until 350 da~3 of :rgc. .Phc I-atio thcii increases until 700 dais. counts
min
Total versus
RI\;A
per
kidnc!
mg
DNA
age in days inuuws
and
RNA
content
IS illustrated
DNA,
l’aster than
in milligrams
in Figure 3. Kidnq though both peak at
I /-. I
I--_._,/’
,.’
--._
-..I
,I’
I’ I’
100
2°C
300
4oc
Age
500
600
(days)
700
800
9
Age changes
in rat liver. kidney
c
/200 Ii00
-
1000
-
900
-
800
-
0
Figure 5 illustrates the incorporation of radiolabeled [14C]thymidine and [‘Hluridine expressed as counts per minute per milligram of DNA and RNA. The incorporation of [liC]thymidine into DNA is highest at 20 days (726 counts,nlin per mg SD 173) and declines to its lowest level at 350 days (247 SD 46) ff < 0.05:. and from there increases until 550 days (488 counts~min per mg SD 248) :P < 0.05;. RNA C3H]uridine incorporation increases from 20 days (205 counts:min per mg SD X0) to 350 d;tys (714 counts,/min per mg SD I 13) [P < 0.05 ). decrelises from 350 days to 700 days (614 countsjmin per mg SD 90). The ratio of [‘“Clthymidinc to [“Hluridine uptake starts high and decreases to its lowest value at 350 days of age. The ratio then changes direction and increases untii 550 days and then decreases again slightly at 700 days.
1
100
.
200
*
.
.
400
300
1
500
Age
600
.
roe
0
*
800900
(days)
F-ig. 3. The plot of liver uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = [2-‘Y‘]thymidine; dashed line = [G3H]uridine; thin solid
line = ratio incorporation
[‘4Cjthymidine/[3H]uridine.
50..
0
1IO5
and spleen
, 100
Figure 6 illustrates total DNA and RNA content (mg) vs age in days of rat spleen. Both total DNA and RNA increase with age until 350 days (DNA 10.3 SD 1.2, RNA 12.8 SD 1.7). The DNA content reaches its highest level at 350 days and then declines until 700 days (6.8 mg SD 1) ;P < 0.05: while the level of RNA shows an increase throughout. In the spleen of 20 and 40 day oid rats the amount of DNA exceeds RNA. However, this situation reverses after 40 days. relative to that of Thymidine incorporation C3H]uridine (Fig. 7) is much higher in all age groups. The highest level of both [‘4C]thymidine and [“Hluridine incorporation is at 20 days (thymidine 2889 countsjmin per mg SD 898. uridine 927 counts: min per mg SD 425) then both values show it drop from 20 to 40 days (thymidine 671 SD 374 {P < O.OSi. uridine 426 SD 164 {P < 0.05)). The ratio
, 200
300
400 Ag8
500
600
700
800
!
idOySi
f-ig. 4. The plot of total kidney DNA and RNA content in milligrams (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = DNA; dashed line = RNA: thtn solid line = ratio DNA/RNA, 0
350 days (RNA 23.5 mg SD 3.3, DNA Il.3 mg SD 1.7). After 350 days. both kidney RNA and DNA shows a slow progressive decline to a lower level at 700 days (RNA 18.9 mg SD 2.5, DNA 8.2 mg SD 2.3). The ratio of DNA to RNA is highest at 20 days, and increases again at 220 days, to a level which remains constant until 700 days.
,
,
,
,
,
,
,
,
100
200
300
400
500
600
700
8009
Age
(days)
Fig. 5. The plot of kidney uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = [2-“%]thymidine; dashed line = [6-3H]uridine; thin solid line = ratio incorporation [‘JC]thymidine/[3H]uridine.
LIIIGI Mtsww~
a IO0
200
300
400 Age
500
600
700
800
(do&)
Fig. 6. The ploi of total spleen DNA and RNA content in miihgrams (left ordinate) and their ratios (right ordinate) vs rat age in days. Solid line = DNA: dashed line = RNA: thin solid line = ratio DNA’RNA.
of [‘~C]thymid~ne~[3H]uridine shows that the drop in [3H]uridine relative to [“%Z]thymidine is larger (Fig. 7). DISCC’SSION It has heen shown that in the liver there is a relationship hetueen the age of an animal and the transformation of hepatocytes from u diploid to polyploid state. S\n.artz (1956) postulated that this transf~~rnl~lti~~llis an increasing function during the first 20 yr of life in humans. Van Bezooijen et td. (1972) found that in rat the production of the polyploid state in liver peaked at about one year. When one compares the life cxpcctancy of rats and humans ntf see that the polyploid state occurs during the equivalent ln~itur~~tion age. Correlation of the results of this study with the above data leads us to the hypothesis that the production of a polyploid state is possibly related to dramatic lowering of [‘“Clthymidine incorporation (Fig. 3) in the liver. In early life, there are potentially a greater number of liver cells able to undergo polyploidy. This means higher DNA synthesis and [‘?Jthymidinc incorporation. In older animals, there would be fewer liver cells able to undergo polyploidy. This would be rellectcd in the lowering of the [“%I’]thymidine incorporation rate. Another explanation for some of the initial lowering in [‘JC]thymidine may be the observation of (Greengard (‘t tri.. 1972) that production of perinatal hematopoetic tissue in liver stops soon after birth. This decrcasc coincides with the decrease in DNA: RNA ratio between 20- to 40-day-old rats (Fig. 2) and also with the decrease in [‘“C]thymidine [“Hluridine ratio during this same period (Fig. 3).
c’t trl.
It is of interest that the total quantity of liver DNA shows an increase while the rate of [‘~C]thyrn~dilie incorporation is fatling. This may mean that 21 control point early in life sets the developmental program of the liver. The [“Hluridine uptake. unlike that of [‘4C]thymidine, reaches a peak at 200 days and consequently decreases to 700 days (Fig. 3). Since the total RNA content shows an increase. the catabolic rate for RNA destruction may be decreasing. The kidney (Fig. 4) shows that total DNA relative to RNA is highest at 20 days. This reflects ;I high nuclear to cytoplasmic ratio. As an animal matures. total DNA doubles by 90 days, while total RNA quadruples. From 90 to 220 days. kidney DNA shows a lower rate of increase. while RNA declines. The drop in total RNA. from 90 days to 220 days, may be a rcsponse to sexual m~~tur~ltion and may reflect an increase in RNA catabolism since synthesis increases (Fig. 4). After 350 days the total content of DNA and RNA, in kidneys (Fig. 4) drops by about lo”,,. The decline of RNA and DNA during this time span was statistically significant j P < 0.05 j and follows the observed decrease in total kidney weight. In the human kidney, maximum size. 270 g, is reached by the third to fourth decade and declines to I85 g by ninth (Epstein. 1979). This loss is mainly cortical and is associated with renal vascular changes. sclerosis and a collapse of the glomerular tuft. The increase in DNA synthesis from 350 to 700 days may be a result of fibroblast proliferation due to autoimmune related damage or a compensatory response to glomerular loss. The spleen is composed of a heterogenous mixture of cells. The initial increase in total RNA content (Fig. 6) ma! correspond with attainment of immunocompe-
-e
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I00 200 300 400 500 600 100 800 900° Age (days) Fig. 7. The plot of spleen uptake in cpm (counts per minute) per milligram of DNA and RNA (left ordinate) and their ratios (right ordinate) vs rat age in daqs. Solid line = [2-“Vlthymidine: dashed line = [6-“Hluridine: thin solid lint = ratio incorporation ~‘~C]thymidine,C’H]uridlnc.
Age
I107
changes in rat Ii\er. kidney and spleen
tence in the RNA-rich plasma cell line. The relative decrease in [“Hluridine incorporation (Fig. 6) implies a negative control point during the neonatal period that ultimately sets the level of total RNA at maturity (Fig. 6). At 90 days (Fig. 7). [‘4C]thymidine incorporation decreases. possibly due to sexual development. The decrease in total quantity of DNA (Fig. 6) after 350 days is statistically significant [P < 0.05), and may express a generalized decline in immunocompetence with age. The increased [“‘Clthymidine incorporation after 350 days may reflect a compensatory response to the drop in total DNA. Thymidine incorporation relative to that of [-‘Hluridine (Fig. 7) is much higher at all age groups. The highest level of both [‘4C]thymidine and [ ‘Hluridine incorporation is at 20 days. Both values show a drop from 20 to 40 days. The ratio of [“Clthymidine [“Hluridine shows that the drop in [ ‘Hluridine relative to [14C]thymidine is larger (Fig. 71. Total DNA and RNA ratio show an almost contenuous decrease (Fig. 6). These data support the view that the ratio of nucleus (DNA) to cytoplasm (RNA) decreases with differentiation and perhaps aging (Minot. 1907).
We would like to submit the following conclusions as a result of our studies. Total DNA increases up to 350 days of age in the liver. kidney and spleen, and the rate of incorporation ol’thymidine decreases from birth to 350 days of age. In the life span of rats. the period corresponding to 350 days of age seems to be a turning point where total DNA per organ declines; paradoxically, the rates of thymidine incorporation increases. This may be an attempt by the organism to re-establish the equilibrium of maturity.
Similarly, the total amount of RNA in liver, kidney and spleen increases from birth to 350 days of age. However, the pattern of uridine incorporation in each organ is different. In the liver. the rate of uridine incorporation increases up to 350 days of age and thereafter decreases. In the kidney, uridine incorporation increases up to 350 days of age and then remains constant. This ma] be the result of a compensatory incrcasc in activity related to the loss of glomeruli. In the spleen. the rate of uridine incorporation is constant after the first 40 days of life.
REFERENCES
Geigy. Scicwtific Ttrhlrs. 5th edn. p. 39. K~qer. New York (1959). EPSTEIN M. (1979) Etfects of aging on the kidney. F&Z Proc. 38. 168-171. GK~ENGARI) 0.. FEIXRMAE;M. & Knox W. F. (1972) Cvtomorphometry of developing rilt liver and Its npplica;ion to enzymic diflerentintion. J. w/l. Biol. 52. 761 272. MFSSINW L. (1972) Interferences in deoxyribose detcrmination by the cysteine-HCI sulphuric acid method /,lt. J. Biwhw. 3, 53 I 536. MESSI~*;FO L. & D’AMI(.o J. (1972) A test for ribose determination without interference from dcoxyribose. Irlr. J. BioclIr/?l. 3. 35 I-356. MINOTC. S. (1907) The problem of age, gro\%th and death fop. Sci. hfon. 71. June 481 496: August 97 120: December 509- 523. SW~RTZ F. J. (I 956) The development in the human hver of multlple desoxyrlbose nucleic acid (DNA) cI;lsses and their relationship to the age of the individual. C/I~I~IOSOU,~,8, 53-72. VAN BEZOOIJANC. F. A.. DELEEW-ISRAEL F. R. & HOLLAXER C. F. (1972) On the role of hepatic cell polyploidy in changes in liver function with age and following partial hepatectomy. Mrcl~. Acqeirq LIrr. 1. ?I 356. Docu~mwta