DTIC ADA630520: Oxidative Stress Precedes Circulatory Failure Induced by 35-GHz Microwave Heating

9 Pages·5.6 MB·English

by  Defense Technical Information Center

SHOCK, Vol. 13, No. 1, pp. 52-59, 2000
OXIDATIVE STRESS PRECEDES CIRCULATORY FAILURE INDUCED BY
35-GHZ MICROWAVE HEATING
John Kalns,*t Kathy L. Ryan,*§ Patrick A. Mason,** John G. Bruno, :til
Robert Gooden,t and Johnathan L. Kiel*
•veridian Engineering, Inc. at Brooks AFB, Texas 78235, tDavis Hyperbaric Laboratory, San Antonio,
Texas 78235; :tAir Force Research Laboratory, Directed Energy Bioeffects Division, Brooks AFB, Texas
78235; §Department of Biology, Trinity University, San Antonio, Texas 78212; 11University of Texas Health
Science Center, San Antonio, Texas 78284
Received 12 Apr 1999; first review completed 26 May 1999; accepted in final form 7 Jul 1999
ABSTRACT- Sustained whole-body exposure of anesthetized rats to 35-GHz radio frequency radiation
produces localized hyperthermia and hypotension, leading to circulatory failure and death.'The physiological
mechanism underlying the induction of circulatory failure by 35-GHz microwave (MW) heating is currently
unknown. We hypothesized that oxidative stress may play a role in the pathophysiology of MW-induced
circulatory failure and examined this question by probing organs for 3-nitrotyrosine {3-NT), a marker of
oxidative stress. Animals exposed to low durations of MW that increased colonic temperature but were
insufficient to produce hypotension showed a 5-to 12-fold increase in 3-NT accumulation in lung, liver, and
plasma proteins relative to the levels observed in control rats that were not exposed to MW. 3-NT accumu
lation in rats exposed to MW of sufficient duration to induce circulatory shock returned to low, baseline levels.
Leukocytes obtained from peripheral blood showed significant accumulation of 3-NT only at exposure levels
associated with circulatory shock. 3-NT was also found in the villus tips and vasculature of intestine and
within the distal tubule of the kidney but not in the irradiated skin of rats with MW-induced circulatory failure.
The relationship between accumulation in liver, lung, and plasma proteins and exposure duration suggests
either that nitro adducts are formed in the first 20 min of exposure and are then cleared or that synthesis of
nitro adducts decreases after the first 20 min of exposure. Taken together, these findings suggest that
oxidative stress occurs in many organs during MW heating. Because nitration occurs after microwave
exposures that are not associated with circulatory collapse, systemic oxidative stress, as evidenced by tissue
accumulation of 3-NT, is not correlated with circulatory failure in this model of shock.
KEYWORDS-nitration, peroxynitrite, 3-nitrotyrosine, radio frequency radiation, microwaves, blood pres
sure
INTRODUCTION duces a pattern of cardiovascular responses similar to that pro
duced by a more familiar modality of heating (i.e., environ
Sustained, whole-body exposure of ketamine-aoesthetized
mental heat stress), de pite a different thermal distribution.
rats to 35-GHz radio frequency radiation produces a pattern of
The identity of primary molecular mediators of MW
heating that is characterized by a pronounced increase in skin
induced circulatory failure is not known. Experimental evi
temperature at the irradiated site with only moderate heating of
dence obtained thus far, however, has shown that some path
the body core ( 1) . This is because the shallow depth of pen
ways are not involved in the pathophysiology of MW-induced
etration at this frequency (35 GHz) results in the deposition of
hypotension and shock. Nitric oxide (NO) is upregulated dur
energy primarily in the cutaneous region (2, 3). Although the
ing shock of many different etiologies and is thought to play an
degree of core body heating differ from that associated with
important role in shock pathogenesis by increasing vasodila
heat stroke induced by envimnmental heating, the cardiovas
tion and thereby contributing significantly to vascular collapse
cular respon es to 35-GHz microwave (MW) heating are re
markably similar to those produced by sustained environmental (5-7). The importance of NO in contributing to many types of
heat stTess (4). That is, both MW heating and environmental experimental shock has been supported by observations dem
heating produce an increase in heart rate. In addition, arterial onst:rating that pharmacological inhibition of de novo NO yn
blood pressure is well maintained during the initial stage of thesis reduces vascular decompensation, increases blood pres
hyperthermia but, as heating continues, blood pressure de sure, and ultimately increase survival (5-7). Similar
creases and eventuates in circulatory failure in both instances. experiments performed in the MW-induced shock model using
Furthermore, pronounced mesenteric va odilation accompa the NO synthase (NOS) inhibitor Nw-nitro-L-arginine methyl
nies the onset of the hypotensive response in both MW heating ester (L-NAME) delivered either chronically during a 2-week
and environmental heat stre s (l, 4). Thus, MW exposure pro- period before MW exposure (8) or at inhibitory doses admin
istered acutely after MW exposure (9) demonstrate that NOS
inhibition has virtually no effect on blood pressure or survival
Address reprint requests to John Kalns, Ph.D., USAFSAM/AFl, 2602 West time. The e findings suggest that de novo synthesis of NO does
G·ue Rd., Brooks AFB, TX 78235. not contribute to MW-induced circulatory failure. Conversely,
52
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Oxidative stress precedes circulatory failure induced by 35-GHz
5b. GRANT NUMBER
microwave heating.
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Kalns J. E., Ryan K. L., Mason P. A., Bruno J. G., Gooden R., Kiel J. L.,
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Institute of Surgical Research, JBSA Fort Sam REPORT NUMBER
Houston, TX 78234
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SHOCK JANUARY 2000 NITRATION AND MW HEATING 53
continuou infusion of S-nitroso-N-acetylpenicillamine a shielded cable placed outside the MW field. All measured variables were
(SNAP), an agent producing pharmacologically significant, recorded continuously throughout experimentation on a Gould TA2000
recorder.
steady- tate levels of NO in vivo, during MW exposure has no Animals were also instrumented to monitor temperature at four sites: I) left
effect on blood pressure or survival time (I 0). subcutaneous (lateral, midthoracic, side facing the MW antenna; T,1); 2) left
Production of highly reactive (relative to NO) and poten tympanic; 3) colonic (5-6 em post-anus); and 4) tail (subcutaneous, dorsal, I
tially toxic-free radical pecies occurs during many forms of em from base). The tail was placed within a 4-cm-diameter tube wrapped with
traumatic injury and shock (11, 12) and an increasingly impor 2C olrapy.e, rMs aorfi ertatdai,o G fAre).q uTehnucsy, trhaed itaaitli owna ss hsiheiledlidnegd mfraotmer idailr e(cBt eekxapeons uSrtee etol MWWire,
tant role for these compounds in shock pathogenesis is emerg yet air wa allowed to now freely around the tail. All temperature measure
ing. In hemorrhagic, endotoxic, and splanchnic artery occlu ments were obtained via thermistor probes (BSD Medical Corporation, Salt
sion/reperfusion models of shock induction, levels of Lake City, UT) attached to a precision thermometry system (model BSD-200;
peroxynitrite, a toxic-free radical formed in situ by the reaction BSD Medical Corporation). All temperature and cardiovascular data were AID
of NO and superoxide, increase within 1 h (13). Furthermore t(oarnianlgo gs yt ot edmig iwtailt)h creoanlv-etirmteed gbrya pahni csIB dMis-pclaoym panatdi bdlaet ac uasntoalmys-idse sciagpnaebdil imtieosn.i
inhibition of peroxynit:rite formation prevents both cellular
damage and vasodilation after hemorrhage or ischemia/ MW equipment
reperfusion (14, 15). Because heme-containing red blood cell Continuous-wave 35-GHz fields were generated by a Millimeter Wave
are reservoirs of NO (16, 17), de novo synthesis may not be a Exposure System (Applied Electromagnetics, Inc., Atlanta, GA). Irradiation
trict requirement for increased levels of free NO if there is was conducted under far-field conditions with the animal centered along the
chemical motivation for liberation. MW exposure may provide wboarse sdiegthetr,m IiIn 0e de amt tihi·eo mex pthoes uarnet esnitnea w. Tithh ea ni necliedcetnrot mpoagwneert idce rnasditiyat ioofn thmeo nfiietoldr
the required chemical motivation for release of NO from red (Narda Microwave Corporation, Hauppauge, NY). The generator power output
blood cells because exposure to 2450-MHz radio frequency was monitored throughout exposure with a model 4-32-B Hewleu-Packard
radiation in vitro has been hown to increase erythrocyte sen power meter. Irradiat-ion wa conducted in an Eccosorb RF-shielded anechoic
sitivity to oxidative hypetthermic hemolysis (I 8) and to revers chamber at the Radiofrequency Radiation Division of the USAF Armstrong
ibly alter the thermodynamics of oxygen binding to hemoglo hLuamboidraittyo ryw,e Brer omokaisn Ataiirn eFdo racte 2 B7.a0s e±, T0e.5x°aCs. aCnhda m20b e±r 5te%m, preersapteucrtei vaenldy , redluartiinvge
bin ( 19). This evidence, albeit indirect, suggests the possibility experiment.ation.
that NO release may occur from red blood cells as they pass
through MW-exposed skin. Experimental procedure
These observations have led us to hypothesize that free radi After surgery, the rat was placed on a Plexiglas holder in an anechoic
cal may act as mediators of the circulatory failure induced by chamber (I). All animals were instrumented with temperature probes (see
sustained MW exposure. Because the in vivo half-life of free AInfstterru ma eIn 0t-amtioinn caonndt rporle ppearriaotido, nt)h ea nadn icmaradl iowvaass ceuxlpaor sleeda disn dfoivr idduataal lyco ilnl ecthtieo nH.
radicals is <l s in vivo (II), we measured the abundance of orientation (left lateral exposure, long axis of body parallel to magnetic field)
3-nitrotyrosine (3-NT), a molecule that is produced from the to 35-GHz radio frequency radiation at an incident power density of 75 mW/
interaction of nitrating free and protein bound tyrosine. We cm2. The orientation of the animal with respect to the field was previously
anticipated that, if free radicals were involved as mediators, ererrsopnoenoseuss ltyo rreapdoirot efdr etqou beencEy (era.gd.i,a tIi)o; nh oatw 3e5v eGr,H czar ddoio vnoast cduilfafer ra nbde ttweemepne rEa taunrde
then 3-NT accumulation would be correlated with shock. In H oricnt.ations (20). This power density results in a whole-body specific ab
other words, the highest levels of 3-NT are expected after sorption rate of 13.0 W/ kg (I).
expo ures associated with shock and that le s accumulation During the I 0-min control period, baseline temperatures were recorded, and
will occur after exposures not a ociated with hock. Tc was maintained at 37.0 ± 0.5°C with use of a water-perfused heating pad.
Rats were randomly assigned to I of? groups (n = 8/group). In the first group,
animals were placed on the MW platform and heated with a water-perfused
MATERIALS AND METHODS heating pad until Tc = 37.0°C; these rats were not exposed to MWs. In this
group, Tc was carefully monitored so that it did not exceed 37.0°C during
ABItinornsontAoaiitmlklu l stAeea Axsnl pi imocre fraaFi lmroH erCeec naaetrl steBh aaan"sndeGd, uaTUindeisemxe a aCfslo oarc mnatdrmhe e wi ptteCreroaeecr eeocd ofua nAnrdediu r c wUFteoesderr ce ae oca fcpR opLerr daoeibvnaoergrcd ah ttob o Lyrty hat beh AoeNr nafanitmto irotaiintelusas"l , eerpiefxir tsepihprsoeressaruc edrtsdiieuav trte(iogMlo yincM)A a. ilWPs I n)od s rd ia sue ecnc.x otirixpnel taethTisrn iecmgud rre eeodtnaou ct apah7t,l 5 etr dhpam irtasosm cspweHpodeeigunrc.er ti,e fP iescMrx.e ptvAReoimPoasteu pswds e oiretlaolfx t puctMheroerenWi tm(in3 nee8uuxn,ne ttt3 sitl94 o i ,n mgd4dere0iocac, uanloipt nerasde r4 t1wuet hrn°eiaCtraitell,,
prepared by the Committee on Care and Usc of Laboratory Animals of the death (8-10). Therefore, we discontinued MW exposure at this point and
dfRnaisrvtdMei trua ptLlleeaa sboStoifpc rL raactagoabrugoieeer-ssaD t(o(aW2rwy6i l leAmxy ni ni2rmg3at taoslx n ,Rw 2Mee0sr.Aoe5 u)ore. cbmTetha)s -einNywe aidwtth ieo frnrweoa mali nt eRdrtie hvsaeiedn aducr aoclfhllooy onC dhioe ousua nvsoceaifdil l. aC inbh lsaetr alaneds agtmhrrbaooliuustgrp ahw,r iretlahrytees d pTweocfe iornaleete ddaa n tttiehansii tsnhs muaebstei snztehetq deou fpae noneditnn dati pnnoosaftilr nycutsim irwsce uanalstsa e tadoac rtbyguu rasotlh luwyop ce 4krw2e . ii7ntnh do ±utT ceOctxi. opI=°no C.s e.4T d2hI n+et o°sa Ce M f,a inWnaaill ;
ltiibmiteu mof. tRhaet exwpeerreim 4e-n5t sm. oAn t1h2s- ohl dli gahntd: 1w2-ehig dhaerdk 3c5y0c-le4 0(0li ggh t( 3o6n7 a±t 6I :g0)0 aat. mth.e) 5th5i sm girno uaps wa atism me acinotnatirnoeld f oart TMcW = e3x7p.o0s u±r e0 .g5r°oCu pasn. dC marodniiotvoaresdcu floarr aa ntdo tatel mo f
was used, and the room temperature was maintained at 22-24°C. perature parameters were continuously recorded during the control period and
throughout the MW or sham exposure period.
Instrumentation and preparation
Animals were ane thetized with urethane ( 1.6 glkg; i.p.), and left and right
ambadionmtaiinnaeld aarte a3s7 .0w e±r e0 .s5h°aCv eddu rbinefgo raell ssuurrggeircya.l Cproolocendicu rteesm. pAe rTaetunroen ( Tcaetl hwetaesr TisImsumee dhiaaterlvy easftte r cessation of MW or sham exposure, the animal was
(PE 20) was placed into a carotid artery for measurement of arterial blood disconnected from all probes and leads and removed from the platform. A
pres. ure. The catheter was attached to a precalibrated blood pressure trans midline incision was made, and a blood sample was withdrawn by cardiac
ducer (model CP-O I; Century, Inglewood, CA). A lead II electrocardiogram puncture into a heparinized vacutainer tube. A 1-mL sample of whole blood
(ECG) was obtained with use of nylon-covered nuorocarbon leads attached to was then immediately added to 9 mL of fixative (FACS lysi11g solution; Bee-
54 SHOCK VoL. 13, No. 1 KALNS ET AL.
ton-Dickson) for processing by flow cytometry. The remaining blood wa exception that nonimmune rabbit polyclonallgG was substituted for the rabbit
separated into pia ma and cellular constituents by centrifugation; plasma wa 3-NT antibody. The concentration of nonimmune IgG was the same as that
collected and immediately frozen and tored at -70°C until time of 3-NT used for 3-NT antibody, and the image location was identical to tl1at used in
enzyme-linked immuno orbent assay (ELISA). After blood collection, samples 3-NT stained section. Tis ue ection exposed to 5 !JoM of peroxynitrite were
of liver, lung, inte tine, kidney, and samples of the abdominal skin from the u eel as positive control , and competitive inhibition of 3-NT antibody binding
left (irradiated) and right (nonirradiated) sides were immediately placed into with free 3-NT (10 !JoM) and reduction of 3-NT before exposure to 3-NT
I 0% neutral-buffered formalin. These were later processed into 4-!Jom sections antibody were u eel as negative controls.
and mounted onto positively charged slides for immunohistochemistry.
Data analysis
Measurement of 3-NT using flow cytometry Mean and standard error of means are shown in graphs. The statistical
After tixation and lysis, cells were wa hed 2 times in 10 mL Dulbecco' significance of differences between control and MW-exposed animals 3-NT
Pho phate-Buffered Saline (DPBS), blocked with 10% normal goat serum in accumulation was determined by using a post hoc Tukey Least Significant
DPBS for 30 min, centrifuged, and then re uspended in I 0 !JoL of rabbit Difference te t. P < 0.05 were considered stati tically ignificant.
polyclonallgG anti-3-NT antibody (Upstate Biotechnology, Lake Placid, NY),
diluted 20-fold in DPBS in 10% normal goat erum, and incubated overnight
RESULTS
in the dark at 4°C. Cells were then washed 2 times with I mL of DPBS, and
the cell pellet wa resuspended in 10 !JoL of phycoerythrin-conjugated goat
Table 1 show the MW exposure times required to obtain
anti-rabbit Fab fragment (Sigma, St. Louis, MO), diluted 20-fold in DPBS, and
either the Tc endpoint or the MAP endpoint (in the 42+ group)
then incubated for I h at room temperature. After incubation, the cells were
washed with I mL of DPBS and then suspended in 400 !JoL of DPBS. in each of the groups. Thermal and cardiovascular responses to
Processing for 3-NT-negative control was identical to the above with the MW exposure in the 42+ group are illustrated in Fig. 1. As
exception that immunoglobulin (lg)G collected from normal rabbit serum noted before (1), Tst began to increase at the onset of MW
(DAKO, Cupertino, CA) was used instead of anti-3-NT lgG antibody. The
exposure, whereas increases in Tc temporally lagged the T
concentration of rabbit lgG in the negative contr I was identical to anti-3-NT 51
increase. Tympanic temperature increa es paralleled those of
antibody. During flow cytometry, cells were selected on a dot-plot of linear
SSC x linear FSC. The fluorescence intensity as ociated with phycoerythrin Tc, whereas tail temperature began to increase (indicating tail
was measured by using the peak height measured for each cell in the FL-2 va odilation) when Tc reached approximately 37.5°C (data not
bandpa s filter. The same instrument settings were used during all run re hown). Heart rate increa ed continuously throughout the pe
ported. The geometric mean of the population of FL-2 signals was computed
riod of MW exposure, reaching the maximum level able to be
with use of Cell Que t software (Becton-Dickinson) and used in subsequent
recorded by our computer system at 42.5 min (Fig. 1). MAP
tatistical comparisons. A minimum of I 0,000 cells was used for population
statistics. was well maintained during the initial phase of MW exposure,
increa ed dramatically to a peak, and then decreased pro
Measurement of 3-NT in plasma foundly. In contra t to previous ob ervations in ketamine- or
A 20-!JoL ample of plasma wa mixed with 80 !JoL of 0.1 M sodium pentobarbital-anesthetized rats (1, 8-10), the decline in MAP
bicarbonate buffer (pH 8.5) and then placed in a 96-well ELISA plate and
in urethane-anesthetized rats in this tudy began as Tc approxi
allowed to bind for 2 h at 37°C and then overnight at 4 °C (21) . The sample was
mated 41.5°C, rather than <40°C. Consequently, the MW ex
then decanted, and the endogenou peroxidase activity neutralized by addition
of 0.3% hydrogen peroxide in methanol for 30 min at room temperature in tJ1e posure time required for circulatory failltl'e (as defined by MAP
dark. Nonspecific binding wa blocked with 10% Normal Goat Serum (NOS; decreasing to 75 mmHg; Table 1) was al o longer than in
BioDesign International, Kennebunk, ME) for I hat 37°C. Wells were washed previous studie in ketam:ine-or pentobarbital-anesthetized rats
3 times with 10% NGS, 0.05% Tween 20 in pho phate-buffered aline (PBS).
(1' 8-10).
After the last wash, 1.6 /Jog of rabbit 3-NT polyclonal antibody (Up tate Bio
Exposure to MW increased 3-NT accumulation in liver, kid
technology) in 100 !JoL of PBS was added to each well for I h at room
temperature, decanted, and washed 3 times with IOo/oNGS/PBS/0.05% Tween ney, lung, intestine, and connective tissue within the gut, and
20. Goat-anti rabbit lgG-biotin conjugate (Sigma) I :2000 in I 0% NOS/PBS also in peripheral leukocytes, but not in irradiated (left abdomi
100 !JoL was added to each well for .I hat 37°C. Wells were washed 2 more nal) skin. Fig. 2 demonstrate that increased fluore cence in
times with 10% NGS/PBS/0.05% Tween 20 followed by I wash with 10%
ten ity, hown as increa ed brightnes , is elevated in livers of
NOS/PBS. A colorometric signal proportional to the amount of 3-NT was
all MW-exposed rats and i negligible in non-MW-exposed
obtained by using an avidin conjugated horseradi h peroxidase system (Vector
Laboratories, Burlingame, CA). After a 20-min development, the reaction was rat . We validated that the antibody was specific to 3-NT by
stopped with addition of 100 !JoL of 2% sodium dodecyl sulfate (SDS) in exposing tissue sections to the 3-NT-·pecific antibody concur
deionized water, and absorbance was read at 405 nm on a microliter plate rently with LO J.LM of 3-NT. This negative control showed
reader.
fluorescence intensity that was similar to that of section ex
posed to the ham antibody control (micrographs not shown).
Immunohistochemical detection of 3-NT In tissue
Furthermore, we exposed tissue sections to the nitrating agent
Tissue sections were deparaffinized in xylene and then rehydrated. Non
specific binding wa blocked by incubation of sections in I 0% NOS in PBS for
I h followed by incubation with the rabbit polyclonal 3-NT antibody I :200 for
TABLE 1. MW exposure times required to attain the T c or MAP
2 h at room temperature. Slides were then wa heel 3 time in PBS and then (I.e., MAP = 75) mm Hg for 42+ group) endpoint in each group
incubated with goat anti-rabbit phycoerythrin conjugate for I h at room tem
perature. Slides were wa heel in PBS, dried, and coverslips mounted with a Tc at endpoint (°C) MW exposure time (min)
antifading agent (Sigma). Images were captured by using an inverted laser
38 15.0 ± 3.0
canning confocal microscope (Carl Zeis , Inc., Thornwood, NY). Excitation
39 24.0 ± 1.5
of phycoerythrin was performed by using a 568-nm laser, and the resulting
40 33.7 ± 1.9
fluorescence was captured by using a 590- to 610 -nrn band-pass filter. The 41 44.2 ± 1.8
intensity of t11e fluorescent signal was measured by using Zeiss Image soft· 42+ 60.3 ± 2.4
ware. Background fluorescence correction was made by subtracting the signal
(42.7 ± 0.1)
obtained from an image prepared in the ame manner a above witJ1 the
SHOCK JANUARY 2000 NITRATION AND MW HEATING 55
46
CONTROL MWON
6
e... 42
~
38
34
44
CONTROL
-. 42
0
0
~ 40
I-
38
60 L---~--------------------------------~
c 640 ,------r-------------------------, FIG. 2. Accumulation of 3-NT in the liver of MW-exposed rats. (a)
~~0~~~--------------~~~-------+ MW (+), 3-NT Ab(+); (b) MW (+), 3-NT Ab(-); (c) MW(-), 3-NT Ab(+); (d)
!~ 420 aMsWso(c-)ia, t3e-dN wT itAhb (p-h)y. cMoaegryntihfircinat.i oPna nxe6l3s0 a, 5a9n0d- bto, 6a1n0d -npman bealsn dc -apnads sd f ialtreer
serial sections, same location within organ.
a:: 360
J:
30-100 L-S _-0 ~5- -10~ -15- -20- 2~5 -3-0 ~35- -40~ 4~5 -5~0 -55- ~6 0 that is statistically indistinguishable from 0. In contra t, the
Time (min) value obtained from animals with Tc = 39°C have a range of
10- 100 with a mean difference of 45.1. A irnilar method was
Tianr5 t~oetF h1lreiGeaf. tl gs1bru.l oobTuocdhpu et apernrmxeepsaooslu suaser nedted; m uHcnpRater,i rld haiMetouaAvrreaPt; s rTc=ac ut e•7 l.ac5 o r mlorenmsic p Htoegnm s(pie.eesr. a,t ottuh rMee W;4 M2 e+Ax Pgp,ro omsueupar)ne. uu emTdhm eian rriaeznleaadtli yoisnni sFhoiigpf. tb4is.e stFuweigese. on4b Ttsahci onawendsd ftrhdoeimf ff eolrluelonnwgce.i ns gi:n 13) -3N-NT Ta ries
present in lung but not liver of control rats; 2) maximum ac
cumulation of 3-NT occurs at Tcs not associated with d rcula
peroxynitrite (5 J,LM) for 2 s and compared these with adjacent tory failure, i.e., Tc < 40°C; and 3) 3-NT accumulation is
sections processed in an identical fashion but exposed to the reduced to control levels at Tc > 40°C.
sham antibody. In this case, staining intensity was significantly
greater in peroxynitrite-exposed ti sue sections stained with the
3-NT antibody. These data show that the procedures that were 400
used here are specific for 3-NT. A similar validation approach
was al o used in flow cytometric determination of 3-NT ac
cumulation in peripheral leukocytes with similar result . 300 Control
~
We also measured and compared the accumulation of 3-NT
~
in liver and lung at various time cotTesponding to Tc vaJues. a:
To realize this goal, micrograph like those hown in Fig. 2 .....
were converted into pixel maps of brightnes or grayscale val ~ 200
ues. In the case of the liver, a grayscale map was constructed .Q0)
for both a 3-NT-stained section and a sham control prepared E
::::s
from the next ti sue section in series. The grayscale pixel maps z 100
from 3-NT immunostained and antibody ham contJ·ol sections
were superimpo ed, and gray cale value of the sham control
were subtracted from the tissue section immunostained with
the 3-NT specific antibody. For example, the grayscale values -20 0 20 40 60 80 1 00
corresponding to Fig. 2b were subtracted from the vaJues for Fluorescence Intensity
Fig. 2a. If there is no accumulation of 3-NT, then the expec
tation is that the mean gray cale difference is 0. Fig. 3 shows FIG. 3. Distribution of 3-NT fluorescence in liver. Relative fluores
the re ults of a typical analysis. In the ease of the temperature cence corrected for background. Sum of 10 images taken from 2 rats {20
control (TC), gray cale differences in pixel values range from images total) at each Tc. Geometric mean of each population is repre
-30 to 30 with the geometric mean occurring at 2.3, a value sented as a symbol in Fig. 2.
56 SHOCK VoL. 13, No. 1 KALNS ET AL.
50
40
t- 30
z
I
20
M
10
TC 37 38 39 40 41 42+
Colonic Temp. (°C)
FIG. 4. 3-NT levels In liver and lungs of MW-exposed rats. Solid
circles represent lung tissue; solid squares liver. Each symbol repre
sents mean of 8-1 0 tissue regions obtained from 2 animals. Values are
in relative fluorescence units.
Fig. 5 how how 3-NT is distributed in various organs after
expo ure to MW. Significant accumulation of 3-NT occurred
within the distal ileum as hown in Fig. Sa. Fig. Sa show that
accumulation was highest in the tips of the villu and on the
endothelium of mall arteries, suggesting that generation of
nitrating compounds occurred directly within these tissues. Fig.
5c bows that accumulation of 3-NT is highest within the cy
toplasm of the distal tubule and is not found within the glo
merulus or within the endothelium lining d1e tubule. Because
d1e tubule is a site of extensive amino acid read orption, we FIG. 5. Distribution of 3-NT In tissue after exposure to MW.
speculate that 3-NT accumulation reflect read orption of free Transmitted light image overlaid with image from 490-to 510-nm band
3-NT from plasma rather than generation of nitrating radicals pass filter corresponding 3-NT. (a) Distal ileum of rat with final Tc =
wid1in the kidney itself. Fig. 5e and Sf show that 3-NT is 42.7•C (i.e., in MW-induced shock). (b) Same region in sham control. (c)
Kidney of rat with final T = 42.7°C. Glomerulus in lower left hand corner;
uniformly distributed throughout the lung and that control ani 0
bright red areas in interstitial spaces between tubules are red blood
mals also show ignificant 3-NT accumulation in the lung, an cells. (d) Sham control. Bright spots are red blood cells; glomerulus
observation d1at is reflected in Fig. 4. shown in upper right hand corner. (e) Lung tissue in animal with final Tc
Accumulation of 3-NT in leukocyte is shown in Fig. 6. Fig. = 39°C (nonshock) showing uniform distribution of 3-NT. (f) Sham con
6 hows d1at, unlike lung and liver, which show maximal ac trol showing positive staining though at only 30% the intensity of MW
exposed rat. Panels c and d, x1000; all others x630.
cumulation at relatively low T S, leukocytes show maximal
0
accumulation after relatively long exposures to MW d1at are
DISCUSSION
ufficient to induce circulatory failure. Bod1 granulated cells
and lymphocytes show similar levels of 3-NT accumulation. The production of nitrating radicals and ubsequent accu
Because lymphocytes have little capacity to generate nitrating mulation of 3-NT occur during models of shock arising from
radicals, it is expected that they will also how little accumu variou physiological insults. Our findings are unique because
lation of 3-NT via autonitration. Thi suggests d1at nitration of MW-induced shock is not as ociated with significant blood
peripheral leukocytes occurred a a result of passage through los or extensive trauma (22). Examination of previous reports
tissue-generating nitrating radicals, not from generation of ni about shock produced by other insults, particularly hemotThage
trating radical themselves. shock, is informative because it demonstrates that MW
Fig. 7 shows that plasma proteins show a pattern of accu induced circulatory failure is not unique for underlying pathol
mulation virtually identical to d1at seen in the lung. Proteins in ogy, but rather shares many common aspects. Szab6 et al. (13)
pla ma are not expected to demonstrate any capacity to gen showed d1at hemorrhage to a blood pressure of 50 mmHg
erate nitrating species d1emselves but rad1er become nitrated produces a 9-fold increase in peroxynill"ite within 1 h, demon-
during passage d1rough ti ue. The observation that 3-NT lev trating that nitrating pecies are generated very rapidly during
el decline at Tcs associated wid1 hock induction ugge ts d1at d1e early phase of shock. Inhibition of de novo NO synthesis by
nitrate groups present on plasma protein , or d1e proteins d1em L-NAME reduced peroxynitrite production to levels ob erved
selves, are rapidly cleared from d1e circulation after rapid pro in control condition (13), suggesting that de novo synthesi i
duction during the first 20 min of expo ure. important in the generation of nitrating radicals during hem-
SHOCK JANUARY 2000 NITRATION AND MW HEATING 57
8 * exposure. In a similar hemorrhagic shock model, Smail et al.
(24) showed that plasma nitrate/nitrite levels increased ap
proximately 4-fold in shock versus nonshock controls 1.5 h
6 after induction and approximately 12-fold at 4 h. Furthermore,
concentrations of nitrate/nitrite at 4 h in liver and intestine were
.... 4 found to be 3- and 4-fold greater, although levels in the kidney
z were not significantly increased. In a model of splanchnic ar
C"I ) 2 tery occlusion shock (25), di tal ileum was necrotic and stained
positively for 3-NT 1 h after reperfusion, a result that i con
sistent with MW exposure that we observed.
0 We observed that oxidative stress, a indicated by accumu
lation of 3-NT in the liver and the lung, occurs after MW
-2 expo ures that are not as ociated with vascular decompensa
37 38 39 40 41 42+ tion and shock, i.e., exposures resulting in Tc < 40°C. Further
Colonic Temp. (°C) more, maximal accumulation occurs at 38-39°C with decline
to near baseline levels as exposure time and Tc increa e further.
pnreeoluasFttuIirGvore.ep 6.h f.liS uls3oo.-r liNeEds aTccc elihrenc vcsleeeyslm s u brnienoipt ls rlyie.s ms tehpneht omlycemyatpenh sov acaynlutdee s no;e fs u5otl-ri8do p rshaqtisuls.a raVefastle urree Msp rWaers eee xnin t Osetxrnpeeso psouinr es ,i tbhrleeess uee lxtopinrlggaan nian t iaoo ncb cfuouilrrd st uhwpis i otphfhi n3e -nNtohTme. e fAnirofsntte r2i 0at phmpartion o xxoiimfd aaMttieWvlye
20 min, production of new adducts may cease, and nitro ad
ducts are removed so that, at longer exposures (and higher Tc),
0.85
- levels of 3-NT in these organs return to baseline levels. In
support of this hypothesis is the finding of Kamisaki et al. (26)
E who showed that 3-nitro adducts to tyrosine in bovine serum
1:
80.75 albumin (BSA) have a half-life of approximately 10 min when
"'':t incubated with rat spleen or lung homogenates. This report also
showed that liver and kidney have virtually no ability to re
tn
.Q move 3-nitro adducts from BSA. Pretreatment of rats with
;<:(- o.s5 endodotoxin increased clearance of 3-nitro adduct , suggesting
z that denitration mechanisms are inducible (26). Because little
I is now known about "denitrase" activity, regulation, or speci
C")
ficity, it is possible that a denitrase enzyme present in the liver
0.55 -t----,---,----r---r-----r---.-----, may have negligible activity toward nitrated BSA but may
37 38 39 40 41 42+ have high activity against nitrated proteins found in the liver
Colonic Temp. (oC) after exposure to MW. The finding that plasma proteins show
a pattern of nitration buildup and decline similar to that ob
FIG. 7. 3-NT levels In plasma. Solid squares represent mean of 6-8 served in lung is supported by the findings of Gow et al. (27)
rats. who showed that nitrated BSA has a half-life of approximately
ooetcitmssqeaItbotnesiniirbthihciuo uortitirrot tlatohci hTechnhntltarddtt lcehusesaiea h ienyftksirnrhtolg(drieaasis a i n moeietfiro m rutpttrcaad cn h hosnNoRef mfroa .one errb re shssdNOyo do tuoCyei heoel cmievdrmtliA cr o oscf vwlafo(eeoot asncir e2)ter icnc eoienc kpis 3nrsolrlsaedmctbsour .eey) tifn oneui u,watraafsn Idhuv errst brsptstNthi eN; tehieh oiolt ioipelfmnaohO nveihnmOioutr s tteciseho ooSn ylsm ehiko,lewt g u slw poee uefraw iisnlbeirwmn ino noon3acuotlvonhhbodgidflu co-cderinie i woN orutrkhbmrunNgfrcart,tdr oi aec hTh h reirtaO tiornet lieeeMh as neelopddiuafn gloa/c rsinrfi rntitcWntwoiaredtsthl cd,ireueooy s l mLta iau 3arrs ortb nnNtst -rris e-u ucio hMfgteiNeoNOixrcneenototnn hden upArduhWgTSdctl ve, aroe nee k uo Mo. aksvsard mm hc rfstr eTuucne aeeeiE tieoxLcogrtnnlorhsdmtoess rinpguhdv-uwic drs oosNmeews,hlbebteo( lt r s sys8eefasnoAuberetruti t g,hi dgyrelsifni rusMmngeta ig a eqed9Muhcpgl ategre,uini)e asEi r ttxni .osiWr teaoitt tcr atiloh anghgclwt beTeeitb o wan-,uonsmhano shoiotc.ahulefotnesu neuoauekfnodlir udug etli ontsd c ehbdgu grc(caent Mkde2a eh tclelt h,aeehtl fv3r e oeaeWlodaiiiiiie)tdn sc rsntfs .r, ssTktfhm1ttgtnfieaneehhohhno5icanmieiinrioe ro tedtvPnr nvsr re mrr mwiuesa eaeaeeictpia c tmirtas ita r ericebnieila aranidcosipo.spnno dittr h gudt,ehlnjoeew dBf iefl ru ue sers twiprssheyNctsl rrnh or peaar.ayciettceio 4mOtiivniretnl,ahatsno eNcr 0ecut. u.i p yc wn kln°irligeetsahtelE r nCnh glee uuooecyspoycei i , ctkln idfn sh un frhteihw eoeytdogns reabea hlroo rcip oncduoiaiaeifyttxgntcchtt ottrohf lhoaert leiiheuiip miedeodyb ndlnnM rmposs ap eli eece rietito,hwunnotnWpintoa i a hb ilvtehortendaailla ae esrte-sloxntu plhwct oer i.y w ciaconnc tahrxos h(aietelvopi dath nm2ti tenppr ldeauvnrry 9erpdadltaareteoceaepei)ss eni rto ,s eo svoi mwtdr l asnaln idnoeitaouonclhcimhab g xlsntn fuetco.seh eos ythd ingl uhuwacs h taTaa n,osttobmicaao nrtrhepz aitctelu lfocdt dee uikhiypr eusmk rpvds liyel eaeenm.teeyathc an rcpseq h r ittmhiti c iuPothlru,iaptomh l(eotsrulseitpaoho2al,aaepnr em orpthreg8netncy oi dwiiglrodep )ttue onoi rahi,d itchchltuc avnfbclheeepaayi eahtt eay.ecy dri3ttltl ran nie loerahe n a- ocv ,dtdusspgm Nlo lhagnem ,ede k hov e.nae iTv nonoinruo anwdynlaeie McE r seti ehudihlu yprds apssvorinaacyitWrtc aarclrcebvtseoaanolhtotiahenys eeeln y f-l
58 SHOCK VoL. 13, No. 1 KALNS ET AL.
phil did show significant levels of 3-NT after exposure as- re ponses to adrenergic agonists (37). Because nitration ha
ociated with shock, and light accumulation of neutrophil been shown to modify or inactivate many proteins, it is also
was observed in colon (not shown). However no neutrophil possible that direct modification of adrenergic receptors, other
were noted in liver and lung after MW exposure not associated receptors, or vascular mooth muscle it elf may leave the vas
with hock, i.e., <40°C. Taken together, these data uggest that culature vulnerable to additional tress. Investigations are cur
activated neutrophil are not re ponsible for the exten ive tis rently underway to establi h if iliere is a direct relationship
sue accumulation of 3-NT ob erved. between nitration and vascular decompensation in iliis model.
It hould be noted that blood pre sure began to decrease in
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