T AD..AO56 536 ARMY PEDICAL ~ESCARCH INS T OF IflCTIOIJS DIStAStS FR—ETC F.’S bfloINDUCTION OF HYPOZINCEPtI* AND HLPATIC METALLOTHIOPICIN SYNTHESIS INai 78 p z SCSOCZNSKI. W .i CANTERBURYUNCLASSIFZ[D P4.

~~~~~~~~ d __

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

I

ILL)~~ C U R IT Y C L A S S I F I C A T I O N OF THIS PAGE (W~,øn Data Entered)

R E1R~~~~~J~~~~ TION P AGE BEFOR E COMPLETING FORM1. RLPO RT NUMB ER .

,,~~~~

f . GOVT ACC ESSION NO. 3. RE CIP IENT ’ S C A T A L O G NUMBER

4. T I T L E (an d Subtfll ...~ 5. TYPE OF REPORT & PERIOD COVER ED

Induction of Hypozincemia and Hepatic -

,.j ~ Metallothionein Synthesis in Hypersensitivity InterimJJ Reactions 6. PERFORMING ORG. REPORT NUMBER

7. AIJTHQR (a) 8. CONTRACT OR GRANT NUMBER (.)

~~~ P. Z. Sobocinski, N. J. Canterbury , Jr.,E. C. Hauer , and F. A. Beau

9. PERFORMING ORGANIZAT ION NAM E A ND ADDRESS 10. PROGRAM ELEMENT. PROJECT , TASKAREA & WORK UNIT NUMBERS

~~ U.S.Army Medical Research Institute ofInfectious Diseases, SGRD—UIP—S 61101A

Fort Detrick, Frederick , MD 21701 3AO611O1A91C 00 138II . CONTROLLING OFFI CE NAME AND ADDRESS 12. REPORT D A T E

U.S.Army Medical Research and Development 19 June 1978Command , Office of the Surgeon General 13. NUMB E R O F PA GES

~~~ Department of the Army. Washington, DC 2fl~ 14 16 + 1 figure

14. MONITORING AGENCY NAME & AODR ESS(I( d i f f e r e n t from Controlling Off Ice) 15. SECURITY CLASS. (of (hi. report)

UnclassifiedI5a . DECLASSIF ICATION/DOWNGRADING

SCHEDULE

16. DISTRIBUTION STATEMENT (of (hi. Report)

Q , Approved for public release — distribution unlimited r~Ir?r~rRnnJ7II~I”l JUL 18 1978

LIJ ~~~ DIS TRIBUTION STATEMENT (of the .b.frec( entered ffl Block 20, if different Iron, Report) IIII _ _ _ _ _ _ _

I fLi... -

‘~‘

E18. SUPPLEMENTARY NOTES

Reprints bearing assigned AD number will be forwarded upon receipt.To be published in PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTA L BIOLOGY ANDMEDICINE.

19. KEY WORDS (Continue on rever.. aid. if n.c. ..ary end identity by block number)

Plasma zincMetallo thionein

Hypersensitivity reactions

\ 2~~. ABST RACT ( mflou. ~~~,.ve,.. -,d It n.c..eat v ~~d iden,i fr by block nun,b.r)

‘ Recent evidence indicates tha t hypersensitivity reactions , produced in rats byLhi .~ a d m in i s t r a t i o n o~ a p ro te in an t igen , alters plasma zinc and iron homuostasis)y depressing concentrations of these trace minerals. Studies were performed todeterm ine if hvpoz incemia occurs as a consequence of redistribution of zinc fromi I . I . ;~~a to L [v ~~r by .t mechanism invo lv ing enhanced hepat ic met a llo th ionein (MT )

n i h e s i s . ~1T. a h igh cvstein—containing cytopiasmic protein , possesses a higha r f i n i t ~or zinc and other heavy metals 1~~ d has been imp lica ted in zinc

DD I~~~ I~Mfl 1d7~3‘ S ....

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L ’ t _ , t ~ I~~~) 1 £ k Lid)

SECURITY CLASSIF ICA TION OF THIS PA GE(ITh.n Data En(.r.d)

~~~‘ ~homeostasis. Antigen challenge (0 .5 mg BSA) in ra ts previously sens i t ized w i t heither 5 or 10 mg BSA produced a significant decrease in plasma zinc and ironconcentrations within 7 hr in an apparent dose—dependent manner. Plasma zincdepression was accompanied by an increase in hepatic MT content as well as MT—associated total Zn and 65Zn used to pulse—label the metalloprotein. Thedepression in plasma zinc,” but not iron, and the enhanced synthesis of MT wassignificantly reduced by prior treatment of rats with actinomycin D. Thisfinding suggests a requirement for new mRNA synthesis for zinc , but not ironalterations during hypersensitivity reactions. Results support the concept thatinduc tion of hepatic MT may be a common mechanism involved in al tered plasmazinc homeostasis regardless of the initiating pathophysiologic condition.

/ ~ ;

us White ~~~~

‘Bu f f SeCtISI 0

W(AIINOUNCED 01~~~~~~

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UNCLASSIFIED

S E C U R I T Y C L A S S I F I C A f l O M OF Pt I $ P*~OE~I4i,en Data En~ere~~

A -~~~~ ~-

Induct ion of Hypozincemia and Hepatic Metallothionein Synthesis

in Hypersensitivity Reactions~—-~.

- - -.-~~~~~~ -- -

VCategory: Pathological Physiology

I

P. Z.1SOBOCINSKI, ~~~. J./ CANTERBURY , JR., E. C. HAUER ,,,~~Ifl~ F. A. BEALL— V — . f —

- / 1 Y~ • I 7~ /I I /__ ._ _ — - - -— —.- .— ./-

United States ~~~~ Medical Research Institute of Infectious Diseases

Fort Detrick, Frederick, Maryland 21701

~ -‘ 7 /, /Running title: METALLOTHIONEINS AND HYPERSENSITIVITY

O~ :‘o ()r 9 //í.~~~~~7.O ~~ A — - .

Mail correspondence to: LTC Philip Z. Sobocinski, Ph.D.Chief, Physical Sciences DivisionUSANRIID, Bldg 1425Fort Detrick, Frederick, MD 21701

2

~~~~~~; 0 11,’ ~‘1;

In conducting the research described in this repor t , the investigators

adhered to the “Guide for the Care and Use of Laboratory Animals” as

promulgated by the Committee on the Revision of the Guide for Laboratory

Animal Facilities and Care of the Ins t i tu te of Laboratory Animal

Resources , Nationa l Research Council . The facili t ies are ful ly

accredited by the American Association for Accreditat ion of Laboratory

Animal Care.

The views of the authors do not purport to reflect the positions of the

Department of the Army or the Department of Defense.

-3

I - —

Antigen challenge of r a t s and rabbits previously sensitized to the

same protein antigen has been demonstrated by Kampschmidt and Pulliam (1)

to induce depressions in plasma zinc and iron concentrations. However ,

these au thors did not determine if the depressions of plasma trace metals

which occur during hypersensi t ivi ty reactions are the result of

redistribution of the metals from plasma to liver. Such redistribution

has been documented in infectious disease (2), inflammation (3), and

endotoxemia (4) .

Our recent investigations into the mechanisms involved in the

hypozincemia of bacterial infections (5), inflammation (6) and endotoxemia

(7) have shown tha t induction of hepatic metallothioneins (MT) in rats is

an integral part of the redis t r ibut ion of zinc from plasma to liver which

occurs in these conditions . Due to their high number of ha i f—cystinyl

residues , MT possesses a rather unique abi l i ty to sequester various

bivalent metals such as z inc , cadmium , and mercury as mercaptide complexes

(8) . Considerable evidence has accumulated which indicates that the

synthesis of hepatic MT is inducible and mediated at the transcriptional

level (9) . These proteins are currently under intensive investigatio n

in an a t t empt to delineate their biological f u n c t i o n ( s ) .

The present study was performed to determine whether zinc accumulates

in the liver during hypersensitivity reactions and if so to what extent

thia is dependent on the induction of hepat-ic MT synthesis . In addition,

the influence of hypersens itivity reactions on the plasma concentrations

of iron was simultaneously assessed .

4

. 1 —

Materials and methods

Male Fisher Dunning rats weighing 234.± 3 g (mean ± SEM) were usedafter a minimum of 1 week acclimation under controlled environmental

conditions as previously described (5). Food (Wayne Lab—Blox , Allied

Mills) and water were provided ad libitum.

Hypersensitivity reactions were produced esaentially by the procedure

described for rats by Kampschmidt and Pulliam (1) except that in one

series of experiments a larger sensitizing dose of protein antigen was

used . Br ie f ly, a solution of bovine serum albumin (BSA , Sigma) in sterile

water was prepared to contain 100 mg/mi and emulsified with an equal

volume of Freund ’s complete adjuvant (Difco). A 0.1 ml aliquot of this

suspension was injected sc into either one or both hind foot pads to

achieve sensitizing doses of 5 and 10 mg respectively. Rats were

challenged 14 days later by ip administration of 0.5 mg BSA dissolved in

sterile water . Control groups included sensitized unchallenged as well

as unsensitized challenged rats.

Blood samples were collected from the pleural cavity 7 hr after the

time of challenge and processed as previously described (5) for the

determinat ion of p lasma zinc (10) and iron (11) concentrations. Livers

were extirpated immediately after exsanguination and processed for the

isolation of MT by the combined procedures described by Sobocinski et al.

(5 ) . These procedures include preparation of liver homegenatc~ . high—speed

ce ntr i f uga t i on , hea t—dena turat ion , acetone f r a c t i o n a t i o n, and sodium

dodccvL suLfa te—polvacrylamide gel electrophoresis (SDS—PAGE) .

~~ ta1lothioneins were labelled in viva (5) with a pulse dose of 65Zn,

10 ~~i/l00 g body weight ( ca r r i e r—free 65Zn, New England Nuclear)

5

—~~- - ~~~~~~~~~ _ _ _

-~~~~~~~~~~~~ - - -- A - - -~~~~~~~ - -

administered ip immediately after the time of challenge. 65Zn—ac tivity in

hepat ic MT f ractions was determined as pr eviously descr ib ed and expr essed

as cpm/ml heated cytosol (5) . Protein content of MT fractions was not

determined since neither biuret nor Lowry procedures y ield valid r esults

(12) . In some experiments relative hepa tic MT content between various

expe rimental groups was estimated by gravimetric analyses of pea k ar eas

obtained by densitometry (Cor ning 740 system) of stained gels a f t er

SDS—PAGE ( 5). Zinc content of MT fract ions was determined by atomic

abso rption spectrophotometry (10) .

In certain experiments , actinomycin D (Calbiochem) pretreatment was

used to inhibit MT synthesis (13) and was administered sc (0.08 mg/l OO g

bod y weight) to sensitized rats 1 hr prior to challenge. Control rats

received an equivalent volume of propy lene glycol used to solubilize the

actinomycin D (5).

D i f f e rences between group means were evaluated for significance by

one—way analysis of variance. A P value < 0.001 was considered significant.

Resul ts

The e f f ec t of sensitization with or wi thout subsequent challenge and

challenge without sensitization on plasma concentrations of zinc and iron

is shown in Table I. Challenge of rats sensitized with 5 mg BSA produced

a significant decrease in zinc and iron concentrations when compared to

values obtai ned in sensitized unchallenged rats or those animals which

received only the standard challenge dose of 0 .5 ng BSA. In addition to

the e f f e c t on p lasma zi nc concentration in sensitized rats , challenge

induced a s i~;n if ican t increase in the Zn and 65Zn content of the hepat ic

MT f ract ion when compared to controls. The e f fec t of sensit izat ion or

6

A :_ — ___ - - -

challenge alone on the zinc content of the MT fraction was similar.

Typical SDS—PAGE separation of the two MT variants, forms A and B

(5), isolated from livers of rats sensitized with 10 mg BSA and challenged

14 days later with 0.5 mg antigen is shown in Fig. 1. Normally, only a

very small amount of MT is detectable in livers of control animals. The

behavior of these MT forms on SDS—PAGE was identical to that previously

described by us for MT isolated from livers of infected rats (5).

In a second series of experiments, a 10 mg sensitizing dose of BSA

and actinomycin D pretreatment was used to demonstrate the inducibility

of MT during hypersensitivity reactions. The effect of sensitization with

the higher dose of BSA and subsequent challenge on plasma zinc and iron

concentrations is shown in Table II. In contrast with the data obtained

with a 5—mg sensitizing dose (Table I), challenge with the same amount of

BSA produced a greater decrease in plasma zinc and iron. Other data

presented in Table II demonstrate that sensitization with 10 mg antigen

rather than 5 mg (Table I) increases the amount of MT—associated zinc (NAZ)

by a factor of 2.4.

Pretreatment of sensitized rats with actinomycin D prior to challenge

significantly reduced MAZ as well as MT content when compared to drug

controls and prevented the hypozincemia associated with challenge in these

rats (Table II). In contrast, actinomycin D pretreatment had no apparent

e f f e c t on the depression of plasma iron induced by hypersensitivity reactions.

Discuss tori

Results obtained in this study have added to our previously published

evidence (5—7) document ing the apparen t fundamenta l importance of hepatic

MT in al tered zinc homeostasis occur r ing in disease states and a f t e r the

7

A — - - - —~ —

~~

adminis t ra t ion of phlog istic agents . Others (14) have recently reported

an increase in hepat ic MT content during stresses which included cold

exposure and strenuous exercise. The induction of hepatic MT is also

known to occur after the administration of various heavy metal salts (15)

and during food restriction (13). It is unknown whether a common FL~mulus

- or several different stimuli exist to explain MT induction in se€~dngly

unrelated stresses.

To explain the hypozincemia and hypoferremia associated with

hypersensitivity reactions, Kampschmid t and Pulliam (1) proposed that

leukocytic endogenous mediator (LEN) is released from phagocytic cells

after stimulation by lymphocytic substance(s). When administered to rats,

LEN has been shown to induce numerous host metabolic responses which

include depressions in plasma zinc and iron concentrations (16). A

similar concep t has been previously used to explain the etiology of fever

during delayed hypersensitivity (17). Evidence presented by Atkins and

Francis (17) suggests that lymphocytes produce lymphokine—like substances

which stimulate phagocytic cells to produce endogenous pyrogen (EP), the

mediator of febrile response. Controversial evidence exists, however ,

concerning the differentiation of LEM and EP (18, 19). Although the

model used in the present studies was originally described as “delayed”

hypersensitivity (1) it has not been established , to the best of our

knowledge, to what extent the measured responses to antigen challenge

involve an t ibody and/or cel l—mediated immune responses . The use of an

ip inj e c t i o n and the rapid responses of serum zinc and hepat ic MT suggest

tha t t~ 11I acute forc iat ion of an t igen—ant ibody comp lexes const i tu ted the

tr i g g er i n g ~t umu lu s . In the absence of conclusive proo f , the involvement

of ~EM in ~he hypo zincemia and hypoferremia of hypersensitivity reactions

8

- — — . - -

remains highly speculative. Although we have shown that LEM administration

to rats can induce MT—like hepatic zinc—bipding proteins (7), we have

been unable to demonstrate a direct effect of LEM on the perfused rat

liver with respect to MT induction (unpublished observations).

In contrast to the proposed role of LEN as a mediator of inflammatory

hvpoferremia (20, 21), recent data presented by Van Snick et al. (22)

indicates that leukocytic lactoferrin mediates plasma iron depression

during inflammatory stress. This evidence together with that obtained

in this study concerning MT and plasma zinc depression suggests a key

role for these two distinct metalloproteins in modulating plasma zinc

and iron homeostasis.

Our observation that actinomycin D pretreatment can significantly

reduce hepatic MT content is compa tible with the concept that new mRNA

is required for enhanced MT synthesis (9, 23, 24). Although it is also

possible that the drug interferes with production of endogenous mediator(s)

such as LEN, no conclusive evidence is available to indicate a mRNA

requirement for the production of potential mediator(s) involved in plasma

zinc depression. The lack of complete inhibition of MT synthesis by

ac tinomy cin D may be due, in part , to the presence of endogenous mRNA

synthesized , but not translated , during the induction of the hypersensitive

state. The dose of actinomycin D used in the present experiments has

hoer~ r~reviously shown to inhibit MT synthesis when administered as early

as 30 mm prior to induction by cadmium (24). Th~ apparen t lack of a

s to ich iometr i c re la t ionship between changes in plasma zinc concentration

and hepatic MT content in drug—treated rats may be attributed to the

effect of actinomvcin D on zinc clearance from blood (13).

9

_____________ - —.

It appears reasonable to conclude that evidence obtained in this

stud y and others (5—7 , 14) indicates tha t -MT induction is a common f ea tu re

of altered plasma zinc homeostasis. To fully understand the pivotal role

of MT in zinc metabolism, the event(s) leading to the initiation of mRNA

synthesis must be further defined .

Summary

Recent evidence indicates that hypersensitivity reactions, produced

in rats by the administration of a protein antigen, alters plasma zinc

and iron homeostasis by depressing concentrations of these trace minerals.

Studies were performed to determine if hypozincemia occurs as a consequence

of redistribu tion of zinc from plasma to liver by a mechanism involving

enhanced hepatic metallothionein (MT) synthesis. MT, a high

cystein—containing cytop lasmic pro tein , possesses a high affinity for zinc

and other heavy metals and has been implicated in zinc homeostasis .

Antigen challenge (0.5 mg BSA) in rats previously sensitized with

either 5 or 10 mg BSA produced a s ign i f i can t decrease in p lasma zinc

and iron concentrations w i th in 7 hr in an apparent dose—dependent manner.

Plasma zinc depression was accompanied by an increase in hepatic MT

content as well as MT—associated total Zn and 65Zn used to pulse—label

the metal loprotein. The depression in plasma zinc , but not iron , and

the enhanced synthesis of MT was s ign i f ican t ly reduced by prior trea tment

of rats with actinomycin 0. This find ing suggests a requirement for new

mRN -\ synthesis for zinc , but no~ iron altera tions during hypersensitivity

reactions. Results support the concept that induction of hepatic MT may

be a comm on mechanism involved in altered plasma zinc homeostasis

r cga rdle,s of the i n i t iat i ng pathophvsiologic condition.

10

—d

Acknowledgments

The technical help of Larry Harris, Jack Simila, Ernie Brueggeinann, and

Karen Bostian, as well as the secretarial aid of R. Arnold is appreciated.

11

a - — -

- — —

—

— — —

References

1. Kampschinid t, R. F., and Pulliam , L. A . , Proc . Soc. Exp . Biol. Med .

147, 242 (1974).

2. Powanda , ‘1. C., Cockerell, G. L., Moe , J. B., Abeles, F. B.,

Pekarek, R. S., and Canonico , P. C., Am. J. Physiol. 229, 479

(1975).

3. Powanda, M. C., Cockerell, C. L., and Pekarek, R. S., Am. J.

Physiol. 225, 399 (1973).

4. Pekarek, R. S., and Beisel, W. R., Appl. Microbiol . 18, 482 (1969).

5. Sobocinski , P. Z . , Canterbury, W. J . , J r . , Mapes , C. A . , Dinterman ,

R. E., Am. J. Physiol. 234, E399 (1978).

6. Sobocinski , P. Z . , Canterbury, U . J., Jr., Mapes, C. A., Fed .

Proc . 37, 890 (1978).

7. Sobocinski, P. Z . , Canterbury , W. J., Jr., and Mapes , C. A. Fed .

Proc . 36, 1100 (1977).

8. Kojima, Y., and K1~gi, J. H. R., Trends Biochem. Sci. 3, 90 (1978).

9. Richards, N. P., and Cousins, R. J., Biochem . Biophys. Res. Commun.

o-~, l.~15 (1975),

10. Pekarek, R. S., Beisel, W. R., Bartelloni , P. J., and Bostian,

K. A., Am. J. Clin. Pathol. 57, 506 (1972).

11. Levy , A. L . , and Vitacca , P . , Clin . Chem. 7 , 241 (1961).

12. Weser , U.. Rupp , H., Donay, F., Linnemann, F., Voelter , W., Voetsch,

U., and lung , C., Eur. .1. Biochem . 39, 127 (1973).

13. Richards , M. P., and Cousins , R. J. , J. Nutr. 106, 1591 (1976).

14. Oh , -~~. J., Deagen , J. T . , Whanger , P. D., and Weswig, P. H.,

A m . J. Physiol. 234, E282 (1978).

L2

- - — — - — —

15. Win ge, D. R., Premakumar , R., and Rajagop alan , K. V ., Arch .

Biochem. Biophys. 170, 242 (1975). -

16. Beisel, W. R., Med . Clin. N. Am . 60, 831 (1976).

17. Atkins, E., and Francis, L., J. Infect. Dis. 128, S277 (1973).

18. Napes, C. A., and Sobocinski, P. Z., Am. J. Physiol. 232, C15 (1977).

19. Merriman, C. R., Pulliam, L. A., and Kampschmid t, R.- F., Proc. Soc .

Exp. Biol. Med . 154, 224 (1977).

20. Kampschmidt , R. F., and Upchurch , H. F., Am. J. Physiol. 216,

1287 (1969).

21. Pekarek, R. S., and Beisel , N. R., Proc. Soc . Exp. Biol. Med. 138,

728 (1971).

22. Van Snick, J. L., Masson , P. L., and Peremans, J. F., J. Exp. Med .

140, 1068 (1974).

23. Squibb , K. S., and Cousins, R. J., Biochem. Biophys. Res. Coinmun.

75 , 806 (1977).

24. Squibb , K. S., and Cousins, R. J., Environ. Physiol. Biochem.

4, 24 (1974).

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5 1 -~~~V C H +1 5 U H Cl ) -El 0-. U I }.4 Cl -1 0 (4 Cl H U U0 (0 Cl 0. H C) U) V 0. ~ .5 (4 5 C(4 (0 V I 0 5 U V Cl V V V V

~ C 5 U Cl .5 i-i 0 ~ - $4 (4H 0 0) 0 Cl) C ~0 U) H Cl) 0)

—I ~ 00 (4 ~ .~4 V — ~-. U—

~-( < 5 0. a 1.-I I -.

- - ci (0 0) V U tO V 0. U --~• - - C-! U) C 0 C H ‘—4 5 V U) C ~ V -s

I “_ ‘ H ~ Cl V U) ‘~~‘ ~~A C)-~~ U 00 Z Cl 4.) U) Cl) )- >.

- -I .0 5 V U -a (4 -~~ ci —. —

I) ‘—‘ U C) 1.-. 4-~ C) V S U ~~-~~ 5 0. 0. 1.1 U U C U

V H C) 1.< Cl V ci

V 0) (4 —, -I (-~ (4 1.1 V Cl) ‘S (U U U

~ 0. U) U) 4.1 Cl) -~~ 5 4.~i14 V C) I V 0.. 1) --4

I _ U) V S S 4.) 5 4 C) S -U H H Ci .—s C1) U 00 00

C C C cc U-4 ci Cl V Cl C >-. ~~1 —

‘ C) _ . —4 — -~ 00 3 > H .0 H Cl) C.) Cl) (0 5a C ..—, 5—. --‘C..

H (‘1 en Ci .0 U V 9.1 00

15

.0 . -

LECENI ) s TO FIGURES -

Figure 1. Typical separation by SDS—gel electrophoresis of A and B forms of

metallothjonejns isolated from livers of BSA—sensitized rats with (left) and

without (right) antigen challenge. Migration was towards anode £ or 1.5 h at

ambient temperature and constant current (2.5 mA/gel). Amount of material

applied to each gel was obtained from equivalent quantities of hepatic cytosol

(see Ref. 5). Material migrating ahead of metallothioneins has not been

identified.

1,

~~~~ -— - - - — -~~

A—w i.

+ _