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THE METABOLIC PROBLEM IN SECONDARY DISEASE
A. L. Kretchmar** and C. C Congdon ***
NOV 1 3 1964
**Medical Division, Oak Ridge Institute of Nuclear Studies
***Biology Division, Oak Ridge National Laboratory

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*Research jointly sponsored by the Medical Division, Oak Ridge Institute of
Nuclear Studies under contract to the U.S. Atomic Energy Commission, and by
the U.S. Atomic Energy Commission under contract with the Union Carbide
Corporation.
THE METABOLIC PROBLEM IN SECOIDARY DISEASE
A. L. Kretchmar and C. C. Congdon
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Highly significant changes in the level of aspartic acid in liver and the
activity of lysozyme (muramidase) in kidney are already detectable 5 and 7 days
after irradiation and treatment with allogeneic bone marrow. There 18 other
evidence of a disordered metabolism of the host particularly in amino acid
metabolic pathways and this disorder 18 already detectable at 5 days and
subsequently appears to spread and lovolve other systems than aspartic acid
metabolism.
We suggest that, talen with the experiments of Lochte et al. (1), our
biochemical results indicate that a disordered host metabolism 18 a very early
consequence of transplantation of allogeneic cells. This 18 presumably the
result of a methotrexate-sensitive component of the grafted cells whose early
activity may lead to secondary disease by injury to an as yet unidentified
target system or systems in the pathways of intermediary metabolism in host
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tissues. Evidence of this injury 18 detectable before the graft has reconstituted
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the hemopoietic system of the chimera, before the dramatic histomathologic
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changes in the lymphoid system are manifest, and before the chimera 18 clinically
(1) Lochte, 8.1., Jr., Levy, A.S., Quenther, D.M., Thomas, E.D., and
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The folloving factors have been suggested as etiologically significant
in homologous disease:
a) graft antihost immunity
b) late Irradiation effects
c) depressed immune competance of the chimera vith chronic lofection
and parasitism
a) possible metabolic defect associated with lack of lymphoid tissue
e) "sump" effect (F48. 1) of proliferating grafted cells
Part of the difficulty in abbiguing priority among these factors 18 the
lack of a generally acceptable characterization of secondary disease. Weight
1088 alone 18 too nonspecific. Dermatologie and visceral lesions are neither
10,72
specific in their histopathology nor waiformly present T1,257; diarrhea,
though prominent in some experiments, 18 absent in others 41,19). Delay of
postirradiation greying, presumably due to impaired hair growth, 18 apparently
a uniform clinical sign but limited to animals with pigmented fur.
The histopathology of the lymphoid tibbue, with proliferation followed
by atropby, appears to be the most central feature of the secondary syndrome,
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but the proliferative reaction in the nodes of the allogeneic chimera must be
distinguished from a proliferative reaction that occurs in animals treated with
syngeneic cells but 18 presumably due to other factors ( 7,8). The atrophic
phase has also been produced by other Kads of experiments; for example, by
thymectomy in newborn mice and by cortisone treatment of newborn mice. And,
Indeed, a syndrome that 18 like secondary disease in radiation chimeras bas
been described in these animals but 18 probably due to infection since the
syndrome was not observed in thymectomized mice maintained germ-free (1, 20).
Thus diminished lymphoid tissue per se does not appear to result in the
secondary syndrome.
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Specific biochemical alterations, if they coud be found, together with
the histopathology of the lymphold tissues might be helpful in giving precision
to the term "secondary disease" and thus make delineation of its biological
parameters possible.
riguro 2 18 a "profile" of the biochemical changes that have so far been
encountered in our study of the metabolic alterations in the secondary syndrome
of Irradiation chimaras. The earliest change in the compounds we have measured
18 la the concentration of free aspartic acid in liver. This 18 already twice
control levels by 5 days after Irradiation; by 7 days, the concentration of
aspartic acid 18 Increased 2.5 times, returning toward normal levels at 9 days
but still olevated by about 50% as late as 35 days after irradiation and bone-
marrow treatment.
Muramidase (Lysozyme) activity in kidney tissue of allogeneic irradiation
chimeras (21,221) 18 also increased at an early time after treatment. Though
normal at 5 days, the activity of this enzyme increases rapidly to about 5 .
times control levels at 9 days, returning toward normal but stiu 1.5 to 2
times control levels at 35 days.
The RNA concentration of liver 18 detectably elevated 9 days after
Irradiation and marrow treatment but nearly normal again by 14 days.
Finally, the concentration of free serine in liver 18 decreased but not
until 14 days, reaching minimum levels of about 1/2 normal concentration on
days 21 and 35.
It is clear froy these results that significant alterations 10
retabolism of the host occur at a very early time, probably as early as
proliferation of the grafted forcign cells and possibly as a consequence of
this proliferation. Discussion of the etiologic factors leading to the
secondary syndrome in Irradiation chimeras must, therefore, include an
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explanation of changes that occur at a stage when tre allogeneic chimera 18
not clinically distinguishable from irradiated animals given syngeneic marrow.
of the factors mentioned previously, the first, sraft antihost imunity, and
the last, a metabolic "sump effect" of the proliferating grafted cells would
seem to be the most likely factors in causing early biochemical changes.
The metabolic sump bypothesis (F48. I) suggests: (a) that the massively
proliferating grafted cells somehow estad.ish metabolic priority over the host
and, (b) that the resultant disorder of the intermediary metabolism in host
tissues is detrimental to the host and possibly eventially to the graft itself.
But extensive proliferation 18 also a characteristic of lofused syngeneio
cells and since the changes, except as noted in Fig. 2, do not oacur in
Irradiated animals given syngeneic celle, some additional factor or factors must
be involved.
Conceivably, the factor could be only quantitative. That 19,
proliferation of cells in an unfavorable (homologous) environment might have to
be more intense or protracted to accomplish a given repopulation of the
hemopoietic system of the irradiated host and, as a consequence, the metabolism
of the host could be taxed to the point of decompensation in one or more of its
intermediary metabolic pathways. The appearance of a syndrome ( 3 ) in
irradiated mice given suboptimal doses of syngeneic cells that has some, at
least, of the features of the secondary disease of irradiation chimeras could
be cited as evidence for this suggestion. The therapeutic effect of injections
of lymphocytes in these animals could be related to the phenomenon described
by Feldman and Yaffe (13,14 ) who found that doses of spleen cells not
ordinarily giving complete protection would do so under conditions of
latense stimulation,
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The evidence 18 extenoive for the first of t'e etiological factors
mentioned. previously, 1.6., graft-anti-host imunity. The possibility that
secondary disease 18 caused by the reverse reactivity (that 18, host anti-graft)
seems unlikely. In the firat few days after irradiation or after sublethal
(5,6,7,26)
doses this roactivity appears to determine the trensplantability, effectiveness,
and long-term survival of the infused foreiga cells. Host anti-graft immunity,
in the first days after treatment at least, would involve injury to relatively
few cells and probably could not seriously disrupt host metabolism. Moreover,
It has been showa ( 13 ) that simultaneous injection of allogeneic and
syngeneic cells leads to the rejection of the foreign cells and recovery of the
Irradiated animal without the complication of secoudary disease. In midlethal
exposures (4,15,23), host anti-graft imunity 18 capable of rejecting an
extensive allogeneic graft, but the animals will, nevertheless, survive 11 they
are given syngene ic marrow cells ( 9 ).
If graft-anti-host Immunity 18 the key factor in the induction of .
secondary disease, then the biochemical evidence presented in Fig. 2 indicates
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that the attack on the host is very early since there is already evidence of
biochemical injury to the host metabolism at days 5 to 9. In addition, we have
previously reported changes in levels of free amino acids in liver (16,17).
Glycine, glutamine, glutathione, B-aminoisobutyric acid and B-alanine, as well
as aspartic acid and serine were alterod. Although these changes bave not all
beon Independent of the strain of mice used in the various experiments, or
have also occurred in the syngeneic bone-marrow treated mice, there is for each
of these amino acids a pattern of change over the interval of 5 to 35 days
after irradiation. Further, the changes in the allogeneic chimeras, when
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compared to syngeneio marrow-treated controls, are quantitatively if not
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qualitatively different; usually more drastic change or fallure to recover to
normal levels was observed. Since the levels of amino acids in the tissue
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pools must reflect the balance between metabolic processes supplying amino
acids to the pool and those tending to remove them, our Pind:Ings indicate that
the metabolism of mice vith grafted allogeneic cells is disordered. A specific
example of this is shown in Fig. 3.
The specific activity of aspartic acid in liver of mice given syngeneic
(IBM) or ellogeneic (HBM) bone marrow 18 compared to that of alanine at 20 to
90 min after injection of uniformly labeled 14c glucose. The reason for
relating the activity of these two amino acids is that the -4c In alanine
during this time, while it changed rapidly ( 17 ), was the same in IBM as in
HBM animals. Thus, although the metabolic conversion of glucose to one anino
acid, alanine, 18 not affected by the presence of grafted allogeneic cells,
conversion to another, aspartic acid, 16 altered.
In parent into F, experiments, Lochte et al. ( 18 ) have shown that
treatment of the mice with methotrexate on days 1, 3, 6, 8 effectively prevents
wasting and secondary mortality. Taken together, our biochemical findings and"
these early treatment experiments suggest the folloring: (a) a disordered host
metabolism 18 a very early consequence of transplantation of allogeneic cells
in irradiated mice and 18 detectable before the graft has reconstituted the
hemopoietic system of the chimera, before the dramatic histopathologic changes
in the lymphoid system are manifest, and before the chimera 18 clinically
distinguishable from its isologous control; (b) a significant factor in the
etiologic complex leading to secondary disease is a methotrexate-sensitive
component of the grafted cells that can be blocked by administration of the
drug during days 1 through 8; (c) the early activity of this component may lead
to secondary disease by injury to an as yet unidentified target system or
systems in the pathways of intermediary metabolism in host tissues; and (a)
this Injury in combination with the metabolic sump effect of a rapidly
proliferating graft may result in an uncompensated and finally irreversible
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disorder in the intermediary metabulism of the host.
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BIBLIOGRAPHY
.
1. Azar, H.A. Bacterial infection and wasting in neonatally thymectomized
rats. Proc. Soc. Exp. Biol. Med. 116
Pr
:
. 2. Balner, A., de Vries, M.J., and van Beldum, D.W. Secoadary disease in rat
radiation chimeras, J. Nat. Cancer Inst. 32: 419, 1964.
• 3. Barnes, D.W.H., Loutit, J.F., and Micklem, H.S. Secondary disease in
lethally irradlated mico restored with synseneic or allogeneic foota)
liver cells.
van Bekkum, D.w. Recovery and therapy of the irradiated organism. In:
Mechanisms of Radiobiology 11: 297, 1960,
• 5. van Bekicum, D.W., and Vos, 0. Immunological aspects of homo-and heterologous
bone marrow transplantation in irradiated animals. J. Cell. and
Comp. Physiol. 50: 139, 1957.
.6. van Bellrum, D.W., Vos, O., and Weyzen, W.W.H. The pathogenesis of the
secondary disease after foreign bone marrow transplantation in
X-irradiated mice. J. Nat, Cancer Inst. 23: 75, 1959.
..
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•7. Congdon, C.C., and Goodman, J.W. Changes in lymphatic tissues during
foreign tissue transplantation, International Symposium on Tissue
Transplantation. pp. 181-207, 1962.
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C
. 8. Congdon, C.C., and Kretar, A.. Increased liver veight in bone marrow
chimeras, Exp. and Molec, Path. 2: 277, 1963.
• 9. Congdon, C.C., Makinodan, T., and Gengozian, N. Effect of injection of rat
bone marrow on recticular tissues of mice exposed to X-radiation in
the midlethal dose range, J. Nat, Cancer Inst. 18: 603, 1957.
. 10. Congdon, C.C., and Urso, I.S. Homologous bone marrow in the treatment of
radiation injury in mice. Am. J. Path. 33: 749, 1957.
• 21. Crouch, B.G., van Putten, L.M.,, van Belum, D.W., and de Vries, M.J.
Treatment of total-body X-irradiated monkeys vith autologous and
homologous bone marrow. J. Nat. Cancer Inst. 27: 53-65, 1961.
.
.
. 12. Denks, J.D., Simmons, E.L., and Wissler, R.W. The histopathology of delayed
death in irradiated mice treated with homologous cells. Radiation
Res. 22.: 557, 1959.
.
.
· 13. Feldman, M., and Yaffe, D. Immunogenetic studies on X-irradiated mice
treated with homologous hematopoietic cells. J. Nat. Cancer Inst, 21:
697, 1958.
· 24. Feldman, M., and Yaffe, D. Immunogenetic studies on X-irradiated mice
treated with hematopoietic cells and grafted with tumor tissues.
J. Nat. Cancer Inst. 23: 109, 1959.
22
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WHI
15. Gengozian, N., and Malinodan, T. Antibody response of lethally X-irradiated
mice treated with rat bone marrow, J. Iliminol. 77: 430, 1956.
• 16. Kretchmar, A.L., and Congdon, c.c. Biochemical changes and increased liver
weight in bone marrow chimeras. Am. J. Physiol. 200: 202, 1961.
• 17. Kretchmar, A.L., and Congdon, C.C. The levels of free aspartic acid,
glycine, and serine in tissues of bone marrow chimeras.
Transplantation 1: 298, 1963.
, 18. Lochte, H.L., Jr., Levy, A.S., Guenther, D.M., Thomas, E.D., and Ferrebee, J.W.
Prevention of delayed foreiga marrow reaction in lethally
irradiated mice by early administration of Methotrexate. Nature 196:
1110-1111, 1962.
. 19. McArthur, W.H., Congdon, C.C., and Kretchmar, A.L. Nitrogen balance in
irradiation chimeras, Proc, Soc. Exp. Biol. Med. In press.
• 20. Miller, J.F.A.P. The thymus and the development of immunologic
responsiveness, Science 144: 1544, 1964.
· 21. Suu, Vu-Thi, Congdon, C.C., and Kretchmar, A.I. Increase in lysozyme
activity in kidneys of irradiation chimeras. Proc. Soc. Exp. Biol.
Med, 213: 481, 1963.
· 22. Suu, Vu-Thi, Congdon, C.C., and Kretchmar, A.L. Lysozyme activity in
radiation chimeras. Proc. Soc. Exp. Biol. Med. 115: 825-829, 1954.
23. Trentin, JoJEffect of x-ray dose on mortality and skin transplantability
in mice receiving F, hybrid warrow, Proc, Soc, Exp. Biol. Med. 93:
98, 1956,
· 24. de Vries, M.J., and Vos, 0. Delayed mortality of radiation chimeras: A
pathological and hematological study. J. Nat. Cancer Inst. 23:
1403, 1959.
. 25. de Vries, M.J., Crouch, B.G., van Putten, L.M., and van Bekisum, D.W.
Pathologic changes in irradiated monkeys treated with bone marrow,
J. Nat. Cancer Inst, 27: 67, 1961.
• 26. Wooles, W.R., and Di Luzio, N.R. Influence of reticuloendothelial
hyperfunction on bone marrow transplantation, Am. J. Physiol, 203:
404, 1962.
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Figure 1. Bypothetical "metabolic sump" effect,
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Pigure 2. "Profile" of biochemical changes in irradiation chimeras. The
bars show levels in mice treated with allogeneic marrow compared
to the uairradiated normal control groups. W is weight of liver,
L is lysozyme (muramidase) activity in kidney, A 18 level of free
aspartic acid in the liver, S 18 level of free serine in liver, and
R 18 ribonucleic acid phosphorus content of liver. These changes
were absent in mice given syngeneic marrow except for liver weight
(ratio 1.3 at 9 days but only 1.1 at 14 days) anâ kidney lysozyme
activity (ratio 1.5 at 9 days but normal after U days).
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Figura 3. Comparison of specific activity (m Ullmicrocuries per millimole)
of carbon of aspartic acid with alanine of liver from Irradiated
mice treated with syngonoio, IRI:, or allogenoio, HBM, bona marrow
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This report WAR preparod as an account of Government sponsored work. Noither the United
Statos, nor the Commission, nor any person acting on behalf of the Commission:
A, Makos any warranty or reprosontation, expressed or implied, with respect to the accu-
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of any information, apparatus, method, or process disclosed in this report may not infringe
privately ownod righta; or
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use of any information, apparatus, method, or process disclosed in this report.
As used in the above, porson acting on behalf of the Commission" includes any em-
ployee or controctor of the Commission, or employee of such contractor, to the extent that
such employee or contractor of tho Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to his employment or contract
ins!th the Commission, or his employment with such contractor.
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