Cerzcer Letters, 5 (1978) 185--189 © E]seviertNorth-HollandScientific Publishers Ltd.
I N F L U E N C F ~ OF T O C O P H E R O L
ON TUMOUR
~85
CELL OXYGENATION
A. K~GERUD, C. KLINTEN]~IERG*,N. LUND** and H.-I. PETERSON Departments o f Surgery L SaM~enska UniversiO~ Hospital, G~teborg, *Oncology and * *Anaesthesiolvgy, UnilJ¢;'sity IJrOspital, Link~$pin8 (Sweden)
(Received22 June 1978) (Accepted 6 July 1978)
SUMMARY Tocophero]l, a well known tissue anti-oxidant, g~ven before local X-ray irradiation o:f 2 transplantable rat turnouts was previously found to increase significantly the effect of irradiation. In the present study tocopherol did not influence the developmenl:, of tumour necroses du~ir~g g r o ~ h of a tra~spl~ntable rat hepatoma. The tumour tissue o×ygenation of a transplantable rat sarcoma was neither :[ound to be influenced by tocopherol. This suggests that an enhanced tumour radic,.~ensitiviW by toeopherol is probably n o t expl~ned by an h~fluence on turn our cell oxygena~,ion.
INTRODUCTION
A decreased turnotLr blood flow rate and a development of central necroses h ~ been observed in :most experimental turnouts [3]. This has been explained either by v ~ c u l a r eorapression through the growing ~umour cell mass [1] or by a shorter turnover tkne oJ[ turnout cells compared 'with vascular endothelia~ cells [8~. Ten to 20% of tumottr cel~s halve been found re, be hypoxic and these cells have also been claimed to be radioresistant [9] In a previous study, the influence of a well known ¢issue ~ntioxidant, tocppherol, or.t the effect of local X-ray irradiation of 2 intramuscularly transplanted rat tumou:m, a 20.methylcholauChrene induced sarcoma and a hepatoma, was investigated [4]. Opposite to what has earlier been suggested [7] a significantly enhanced effect of X-ray irradiation was observed after pretreatment of animal.,~with tocophero!L in a dose o f 5 rag/100 g body wt. The mechanisms behind the enh~ancing effect of tocopherol in local tL'Lmourirradiation might be explained by an influence ,)n turnout cell oxygenation, Tiros, the aim of ~he present study was to [nves~igate the influence of tocopherol on turnout oxygen tension and on the de~1elol~ment of t u m o u r necroses in the previously studied tat turnouts. Address correspondence #~o: ]91'.H.-L Peterson, Laboratory of RheoIogy, Depar~raen[ of
Surgery I, Sahl~re,nskaUniversil;yHospital, S-413 45 G~teborg, Sweden.
J 86 MATERIALS AND METHODS
Anim~Is and tumours Inbred rats from a Lister .,;tnfin of an~nals with a mean body weight of 200 g were studied. In the first experiment a hepa~oma in its 205th transfer generation was transplanted intramuscularly b,y tmchar technique (about 1 mm~ turnout tissue) and in tha second experiment a 20-methylcholanthrene indtXced sarcoma in its 137th t:ransfer generati,an was transplanted by an identical tec!hnique intraparenchyma0y in~o 1 kidney of the anitnals. The lat÷,er transplantation area was chosen h~ ~;he s~udy on turnout oxygenation since it was found to give a free tumour tiss~e surface with a minimum of dissection. Both turnouts were recently described in deSaJl [3].
Tocopherol A DL-a-tocopherol prepaIat:ion for veterinary use (Ido-E Aquosoum vet. v , Ferrosan, Sweden), centah~hlg 50 mg tocopheroi/ml, was injected Latramu~cularly in single or repeated dose~; Xr~:o a normal hindleg of the animals.
Recording of turnour tissue oxygenation For measurements of the oxy¢len tension field of turnout tissue the MDOelectrode was used [2]. This electrode measures oxygen tension in 8 points simult~meously. Wide variations in oxygen tension in tissue depending on the relation of the measulfing point,s to the capillary system can thus be recorded [61. The prep,aratior, of local ti.~sue pOz (PRO2) b y means of a frequency distribution is a suitable method to describe the 3-dimensional tissue oxygen prc~ ~ure field. The fnaquency distribution alIow,~ a comparison of different stak, s .of tissue oxysen supply [5]. EXPERLMENTS
Experirnen~ I Three weeks a~er intramuscular transplanl~ation Jinto 20 animals of the hepatoma, sphe~.cal turnouts ,Nith a mean di~neter of 15 mm were palpated. A.t ~his time the animals welm sac~:ificed ,and tume urs were dissected. From the centr~ parts of the tu~our,~ ;3 tJ~in tape section~ with 10 mm diameter were take~.. Ten anirnals weJre control.,~ and 10 animals were given tocopherc', in a dose of 2.5 mg/t00, g body wt. once a week, starting at the time of tuLlour transplantation. In the 60 tape section,s obtained, necrotic areas could easily be identified by conventional light microscopy. By a mirror system adapted to the microscope, a picture of a tape seq:'.t,J~nwas projected on a paper. Th~ picture was g~aFhically recorded wi~h itls necrotic and non-necrotic areas. These ~xeas were measured p,lan~metrk.,aUy and the quotients between necrotic areas and total section arenas were calculated.
187
Experiment H Sarcoma tissue was transplanted into I kidney in 12 animals. One week later ~ocoph¢~ol was admini~:~ered intramuscularly ~n a single dose of 5 mg/100 g b o d y wt, J~] 6 anin~tals. ' ~ o weeks after t u r n o u t transplantat~.on all animals were lap~zotomized ur~dLerether anaesthesia and the t u m 0 u r surfaces were gently ur~covered. Recordings of t u r n o u t oxygenation were made at 120 different p o i n ~ of ~he surface of each turnout. The frequency distribution of o:.,::;zgen tension walues in control and tocopherol treated animals was calculated for e~mh ani~mI [5]. RESULTS
In the _frst expelSment calculations of ~he q u o t i e n ~ between necrotic turnout areas and tc,tal tz~mcur section areas gave a mean of 0.26 + 0.15 for tocophe~ol t~eated anim~fls and 0.22 -+ 0.10 for control anLimals. The difference calcu]lated by Student's t-test was n o t significant (P > 0.10). In the second experiment freq :ency distributions of oxygen tension values were ,calculated for each animal as exeraplified in Fig. 1. One control animal was excluded due tc, signs of general asphyxia during the experiment. Statist:Lcal calculation did not reve~[ any significant difference between control and toc,)phe~-ol ti:eated animJtls in mean, S.D., skewness or kurtosis (P > 0 . ! 0 accord!ing to the Wilcox:on test) (Table 1.).
DISCUSSION In this study intr~wnuscularadministration of tocophero! had no sJgnificarr~ cell °/o re~ativ~ ¢ount~ freqL.,erlcy
24 .20
'mmHcj Fig. 1. Frequency distribution f~omone tumour (hepatoma), tran~,pLa.tedin ~ ~ ~,~tkMm~,~'. based on 120 re ~dStrationsof ti~ue oxygen tension,
188
E~
0 1.4 z-:~. c:
,4
. ~.. c~ ~.
.¢, 0
O ~ Q ~
N
N
,e~
.--
N,~
~,~
:~.~ °
Z
~
~
e f f e c t o n necrosis d e v e l o p m e n t during t u r n o u t grov~¢h, n o r was t h e tu~r.ovr tissue o x y g e n a t i o n influenced b y t o c o p h e r o l . In a pre~Jous s t u d y [4] o n rats transplant~.~d wiith the same t u m o u c s tocop h e r o l enhanced signffic~mtly t h e e f f e c t o f local X-ray irzadiatio'_~ on the t u m o u r grc,wth rate. T h e observations m a d e in t h e present study" suggests ~th~:~ t h e enhancing effect o f t o c o p h e r o l :in t u r n o u t irrzLdiation i:; n o t ~xpl~ined by a generally i m p r o v e d tumo~ar cell o x y g e n a t i o n . R e c o r d i n g o f t h e d e v e l o p m e n t o f necroti.c areas in turnovers during: grc w ~ b y a conventional histological t e c h n i q u e will give a coarse underst.~nding c):[ t u r n o u t blood flow distribution and o x y g e n a t i o n . However, a re:irked influer~ce o f t o c o p h e r o l on t u r n o u t o x y g e n a t i o n might have been e x p e c t e d to be refilected also b y l~his technique. T h e enhancing effect o f t o c o p h e t o l on t u m o u r irradiation m i g h t still be explained by an influence on radio-resistant hypo~:i!c ~umour ce]:ts, bttt o~her m e c h a n i s m s than an increased t u r n o u t cell o x y g e n a t i o n have t o be searched REFERENCES 1 Folkman,. J. (1974) Turnour ani~iogenesis. Adv. Cancer Res., I9,331--358. 2 ]![essler, 1~i.and Grglnewald, W. (1969) Possibilities el" zl~easurimg oxygen pressure field:~ in tissue by multiwh'e p:latinum electrodes. Prog. Respiir. Res., 3,147--152. 3 Kjart:aa~:on, I. (1976) Tumour circulation. An experimental study in the rat with a comparison of differenl~ methods for estimation of tumour blood flow. Acta Chir. Scand. Suppl., 471. 4 K~gemd, A., Ho~m, G., Larsson, H. and Peterson, H.-I. (1978,) Tocopherol and Local )[-ray Irradiation of two T'tansplantable Rat Turnouts, Cancer Lett~ rs,. 5,123--129. 5 Ltlbbezs, D.W. (1977 ) Quantitative measurement and description of oxygen supply ~co tissue, I~,: Oxygen and Pi~ysiological Function, Editor: F,F. Jobsis. Profe;~sional Information Library, Dallas, U~A.
6 Lund., N., Jozfeldt, L., Lewis, D.H., LSf~triim, J R and 0dinah, S. (1977) Oxygen tension iin muscle tissue of intensive care patients, lntens. Care Med., 3, 124. 7 Sakaxnoto, K. and Sakka, M. (1973) Reduced effect of irradiation on normal ar~dl ma!igna:nt ceils irradiated i:ttvice in mice pre-treated with vitamin E. Br. J. Radiot., 46, 5 3g,'--5'ir'l).
8 ~]Pannoek,J. (1970) Popula¢io~ kinetics of carcinoma cells, capillary endothelial ceils and fibrobkLsts in a transplanted mouse maml~aary carcinoma. Cancer Re,-'..,30, 2470--.'.)474. 9 van Putten, L. and Kalll~an, R. (1968) O.~:ygenatlon status of a transplantable turnout dur'mg fractionated radiiation therapy. J. Natl. Cancer l~ust., 40, 44:1--451.
I N F L U E N C F ~ OF T O C O P H E R O L
ON TUMOUR
~85
CELL OXYGENATION
A. K~GERUD, C. KLINTEN]~IERG*,N. LUND** and H.-I. PETERSON Departments o f Surgery L SaM~enska UniversiO~ Hospital, G~teborg, *Oncology and * *Anaesthesiolvgy, UnilJ¢;'sity IJrOspital, Link~$pin8 (Sweden)
(Received22 June 1978) (Accepted 6 July 1978)
SUMMARY Tocophero]l, a well known tissue anti-oxidant, g~ven before local X-ray irradiation o:f 2 transplantable rat turnouts was previously found to increase significantly the effect of irradiation. In the present study tocopherol did not influence the developmenl:, of tumour necroses du~ir~g g r o ~ h of a tra~spl~ntable rat hepatoma. The tumour tissue o×ygenation of a transplantable rat sarcoma was neither :[ound to be influenced by tocopherol. This suggests that an enhanced tumour radic,.~ensitiviW by toeopherol is probably n o t expl~ned by an h~fluence on turn our cell oxygena~,ion.
INTRODUCTION
A decreased turnotLr blood flow rate and a development of central necroses h ~ been observed in :most experimental turnouts [3]. This has been explained either by v ~ c u l a r eorapression through the growing ~umour cell mass [1] or by a shorter turnover tkne oJ[ turnout cells compared 'with vascular endothelia~ cells [8~. Ten to 20% of tumottr cel~s halve been found re, be hypoxic and these cells have also been claimed to be radioresistant [9] In a previous study, the influence of a well known ¢issue ~ntioxidant, tocppherol, or.t the effect of local X-ray irradiation of 2 intramuscularly transplanted rat tumou:m, a 20.methylcholauChrene induced sarcoma and a hepatoma, was investigated [4]. Opposite to what has earlier been suggested [7] a significantly enhanced effect of X-ray irradiation was observed after pretreatment of animal.,~with tocophero!L in a dose o f 5 rag/100 g body wt. The mechanisms behind the enh~ancing effect of tocopherol in local tL'Lmourirradiation might be explained by an influence ,)n turnout cell oxygenation, Tiros, the aim of ~he present study was to [nves~igate the influence of tocopherol on turnout oxygen tension and on the de~1elol~ment of t u m o u r necroses in the previously studied tat turnouts. Address correspondence #~o: ]91'.H.-L Peterson, Laboratory of RheoIogy, Depar~raen[ of
Surgery I, Sahl~re,nskaUniversil;yHospital, S-413 45 G~teborg, Sweden.
J 86 MATERIALS AND METHODS
Anim~Is and tumours Inbred rats from a Lister .,;tnfin of an~nals with a mean body weight of 200 g were studied. In the first experiment a hepa~oma in its 205th transfer generation was transplanted intramuscularly b,y tmchar technique (about 1 mm~ turnout tissue) and in tha second experiment a 20-methylcholanthrene indtXced sarcoma in its 137th t:ransfer generati,an was transplanted by an identical tec!hnique intraparenchyma0y in~o 1 kidney of the anitnals. The lat÷,er transplantation area was chosen h~ ~;he s~udy on turnout oxygenation since it was found to give a free tumour tiss~e surface with a minimum of dissection. Both turnouts were recently described in deSaJl [3].
Tocopherol A DL-a-tocopherol prepaIat:ion for veterinary use (Ido-E Aquosoum vet. v , Ferrosan, Sweden), centah~hlg 50 mg tocopheroi/ml, was injected Latramu~cularly in single or repeated dose~; Xr~:o a normal hindleg of the animals.
Recording of turnour tissue oxygenation For measurements of the oxy¢len tension field of turnout tissue the MDOelectrode was used [2]. This electrode measures oxygen tension in 8 points simult~meously. Wide variations in oxygen tension in tissue depending on the relation of the measulfing point,s to the capillary system can thus be recorded [61. The prep,aratior, of local ti.~sue pOz (PRO2) b y means of a frequency distribution is a suitable method to describe the 3-dimensional tissue oxygen prc~ ~ure field. The fnaquency distribution alIow,~ a comparison of different stak, s .of tissue oxysen supply [5]. EXPERLMENTS
Experirnen~ I Three weeks a~er intramuscular transplanl~ation Jinto 20 animals of the hepatoma, sphe~.cal turnouts ,Nith a mean di~neter of 15 mm were palpated. A.t ~his time the animals welm sac~:ificed ,and tume urs were dissected. From the centr~ parts of the tu~our,~ ;3 tJ~in tape section~ with 10 mm diameter were take~.. Ten anirnals weJre control.,~ and 10 animals were given tocopherc', in a dose of 2.5 mg/t00, g body wt. once a week, starting at the time of tuLlour transplantation. In the 60 tape section,s obtained, necrotic areas could easily be identified by conventional light microscopy. By a mirror system adapted to the microscope, a picture of a tape seq:'.t,J~nwas projected on a paper. Th~ picture was g~aFhically recorded wi~h itls necrotic and non-necrotic areas. These ~xeas were measured p,lan~metrk.,aUy and the quotients between necrotic areas and total section arenas were calculated.
187
Experiment H Sarcoma tissue was transplanted into I kidney in 12 animals. One week later ~ocoph¢~ol was admini~:~ered intramuscularly ~n a single dose of 5 mg/100 g b o d y wt, J~] 6 anin~tals. ' ~ o weeks after t u r n o u t transplantat~.on all animals were lap~zotomized ur~dLerether anaesthesia and the t u m 0 u r surfaces were gently ur~covered. Recordings of t u r n o u t oxygenation were made at 120 different p o i n ~ of ~he surface of each turnout. The frequency distribution of o:.,::;zgen tension walues in control and tocopherol treated animals was calculated for e~mh ani~mI [5]. RESULTS
In the _frst expelSment calculations of ~he q u o t i e n ~ between necrotic turnout areas and tc,tal tz~mcur section areas gave a mean of 0.26 + 0.15 for tocophe~ol t~eated anim~fls and 0.22 -+ 0.10 for control anLimals. The difference calcu]lated by Student's t-test was n o t significant (P > 0.10). In the second experiment freq :ency distributions of oxygen tension values were ,calculated for each animal as exeraplified in Fig. 1. One control animal was excluded due tc, signs of general asphyxia during the experiment. Statist:Lcal calculation did not reve~[ any significant difference between control and toc,)phe~-ol ti:eated animJtls in mean, S.D., skewness or kurtosis (P > 0 . ! 0 accord!ing to the Wilcox:on test) (Table 1.).
DISCUSSION In this study intr~wnuscularadministration of tocophero! had no sJgnificarr~ cell °/o re~ativ~ ¢ount~ freqL.,erlcy
24 .20
'mmHcj Fig. 1. Frequency distribution f~omone tumour (hepatoma), tran~,pLa.tedin ~ ~ ~,~tkMm~,~'. based on 120 re ~dStrationsof ti~ue oxygen tension,
188
E~
0 1.4 z-:~. c:
,4
. ~.. c~ ~.
.¢, 0
O ~ Q ~
N
N
,e~
.--
N,~
~,~
:~.~ °
Z
~
~
e f f e c t o n necrosis d e v e l o p m e n t during t u r n o u t grov~¢h, n o r was t h e tu~r.ovr tissue o x y g e n a t i o n influenced b y t o c o p h e r o l . In a pre~Jous s t u d y [4] o n rats transplant~.~d wiith the same t u m o u c s tocop h e r o l enhanced signffic~mtly t h e e f f e c t o f local X-ray irzadiatio'_~ on the t u m o u r grc,wth rate. T h e observations m a d e in t h e present study" suggests ~th~:~ t h e enhancing effect o f t o c o p h e r o l :in t u r n o u t irrzLdiation i:; n o t ~xpl~ined by a generally i m p r o v e d tumo~ar cell o x y g e n a t i o n . R e c o r d i n g o f t h e d e v e l o p m e n t o f necroti.c areas in turnovers during: grc w ~ b y a conventional histological t e c h n i q u e will give a coarse underst.~nding c):[ t u r n o u t blood flow distribution and o x y g e n a t i o n . However, a re:irked influer~ce o f t o c o p h e r o l on t u r n o u t o x y g e n a t i o n might have been e x p e c t e d to be refilected also b y l~his technique. T h e enhancing effect o f t o c o p h e t o l on t u m o u r irradiation m i g h t still be explained by an influence on radio-resistant hypo~:i!c ~umour ce]:ts, bttt o~her m e c h a n i s m s than an increased t u r n o u t cell o x y g e n a t i o n have t o be searched REFERENCES 1 Folkman,. J. (1974) Turnour ani~iogenesis. Adv. Cancer Res., I9,331--358. 2 ]![essler, 1~i.and Grglnewald, W. (1969) Possibilities el" zl~easurimg oxygen pressure field:~ in tissue by multiwh'e p:latinum electrodes. Prog. Respiir. Res., 3,147--152. 3 Kjart:aa~:on, I. (1976) Tumour circulation. An experimental study in the rat with a comparison of differenl~ methods for estimation of tumour blood flow. Acta Chir. Scand. Suppl., 471. 4 K~gemd, A., Ho~m, G., Larsson, H. and Peterson, H.-I. (1978,) Tocopherol and Local )[-ray Irradiation of two T'tansplantable Rat Turnouts, Cancer Lett~ rs,. 5,123--129. 5 Ltlbbezs, D.W. (1977 ) Quantitative measurement and description of oxygen supply ~co tissue, I~,: Oxygen and Pi~ysiological Function, Editor: F,F. Jobsis. Profe;~sional Information Library, Dallas, U~A.
6 Lund., N., Jozfeldt, L., Lewis, D.H., LSf~triim, J R and 0dinah, S. (1977) Oxygen tension iin muscle tissue of intensive care patients, lntens. Care Med., 3, 124. 7 Sakaxnoto, K. and Sakka, M. (1973) Reduced effect of irradiation on normal ar~dl ma!igna:nt ceils irradiated i:ttvice in mice pre-treated with vitamin E. Br. J. Radiot., 46, 5 3g,'--5'ir'l).
8 ~]Pannoek,J. (1970) Popula¢io~ kinetics of carcinoma cells, capillary endothelial ceils and fibrobkLsts in a transplanted mouse maml~aary carcinoma. Cancer Re,-'..,30, 2470--.'.)474. 9 van Putten, L. and Kalll~an, R. (1968) O.~:ygenatlon status of a transplantable turnout dur'mg fractionated radiiation therapy. J. Natl. Cancer l~ust., 40, 44:1--451.
