Br. J. Caner (I 992), 66, 297 33 '." Mamillan Press Ltd., 1992 Br. J. Caner (1992), 66, 297-33 Mamillan Press The effet of omega-6 and omega-3 fatty aids on 3H-thymidine inorporation in hepatoma 7288CTC perfused in situ L.A. Sauer & R.T. Dauhy Caner Researh Laboratory, Medial Researh Institute, The Mary Imogene Bassett Hospital, Cooperstown, New York 13326, USA. Summary Ingestion of diets ontaining orn oil or marine fish oils is known to inrease or derease, respetively, the growth of transplantable rodent tumours. The ative agents in these oils have been identified as linolei aid (in orn oil) and omega-3 fatty aids (in marine oils), but it is still not known how they influene the tumour growth proesses. In these experiments we examined the effets of plasma free omega-6 and omega-3 fatty aids on the rate of 3H-thymidine inorporation in tissue-isolated hepatoma 7288CTC perfused in situ. Host Buffalo rats were fed an essential fatty aid-defiient diet. Plasma and tumours in these animals ontained low endogenous levels of both omega-6 and omega-3 fatty aids. Perfusion of these tumours for 2 h with donor whole blood ontaining added omega-6 free fatty aids, inluding.5 mm linolei (C18:2,N-6), gamma-linoleni (C18:3,N-6), dihomo-gamma-linoleni (C2:3,N-6) or arahidoni aids (C2: 4,N-6), inreased the rate of 3H-thymidine inorporation. Linolei aid was about three times more effetive than the other omega-6 fatty aids. Typial hyperboli substrate-saturation urves were observed as the plasma free linoleate or arahidonate onentration was inreased. When perfused alone plasma free omega-3 fatty aids had no effet on tumour 3H-thymidine inorporation, but in the presene of linolei aid the omega-3 fatty aids, alpha-linoleni (C18:3,N-3) and eiosapentaenoi (C2:5,N-3), ompetitively inhibited both tumour linoleate uptake and the stimulative effet on 3H-thymidine inorporation. The results suggest that the ambient plasma free linolei and arahidoni aid onentrations in host arterial blood diretly influene the rate of tumour DNA synthesis. Plasma free omega-3 fatty aids appear to modulate the effet of linolei aid by ompetitively inhibiting its uptake. These relationships ould explain the ations of dietary linolei and omega-3 fatty aids on tumour growth in vivo. Dietary fats plays an important role in tumour growth in rodents. High fat diets ontaining orn, soybean or safflower oils derease the latent period between implantation and tumour appearane (Rogers & Wetsel, 1981; Abraham & Hillyard, 1983) and inrease the rate of growth of established tumours (Rogers & Wetsel, 1981; Gabor et al., 1985). The searh for the substane responsible for the growth-stimulative effet has foused on linolei aid (C18:2,N-6), an essential fatty aid that onstitutes 55 to 8% of the fatty aid ontent of these oils. Feeding mie a fat-free diet supplemented with as little as.1% purified linolei aid inreased growth of a transplantable mammary tumour as muh as a diet ontaining 15% orn oil (Hillyard & Abraham, 1979). Surprisingly, a fat-free diet supplemented with arahidoni aid (C2:4,N-6) alone had no signifiant effet on tumour growth (Hillyard & Abraham, 1979; Hillyard et al., 198). Measurements of tumour growth in groups of rats fed diets ontaining different amounts of orn oil showed inreased growth as the linolei aid ontent was inreased; a plateau was reahed at about 3 to 4% orn oil (Ip et al., 1985) suggesting that the effet of linoleate was saturable. Dietary fish oils, on the other hand, have effets opposite from those of orn oil. Ingestion of fish oils inreased the length of the latent period between implantation and detetion of a palpable tumour (Jurkowski & Cave, 1985) and slowed the growth rate of established tumours (Karmali et al., 1984; Gabor & Abraham, 1986). Addition of fish oils or individual omega-3 fatty aids to diets ontaining linolei aid inhibited the stimulative effet of the linoleate on tumour growth (Gabor & Abraham, 1986). In previous experiments, using tissue-isolated hepatomas 7288CTC perfused in situ, we identified plasma free linolei and arahidoni aids as the ative agents in hyperlipemi blood responsible for a stimulative effet on tumour 3Hthymidine inorporation (Sauer & Dauhy, 1988). Subsequently, we showed that the uptake of these free fatty aids by hepatoma 7288CTC in vivo was dependent on the rate of supply to the tumour (Sauer & Dauhy, 1992). In this study we have used tissue-isolated tumours perfused in situ to examine the effets of purified omega-6 and/or omega-3 fatty Correspondene: L.A. Sauer. Reeived February 1992; and in revised form 21 April 1992. aids on the inorporation of 3H-thymidine into tumour DNA. Materials and methods Reagents Linolei, arahidoni, gamma-linoleni (C18:3,N-6) dihomogamma-linoleni (C2: 3,N-6), alpha-linoleni (C18: 3,N-3), otadeatetraenoi (C18 :4,N-3), eiosapentaenoi (C2: 5,N- 3) and doosahexaenoi aids (C22:6,N-3) were purhased from Sigma Chemial Co., St. Louis, MO. The purity of these fatty aids was measured by gas hromatography and was greater than 98%, in agreement with the speifiations of the supplier. Heptane (HPLC grade), hloroform, methanol and ethanol were obtained from Fisher Chemial Co. The heptane and hloroform were redistilled before use. Methyl esters of rapeseed oil fatty aids and other standard free fatty aids were purhased from Supelo, Bellefonte, PA. [Methyl- 3H]Thymidine (6.7 Ci mmol-1) was a produt of Researh Produts International, Mt. Prospet, IL. [1-_4C]Linolei aid (5mCimmol-1) was purhased from NEN Researh Produts, Boston, MA. Animals and diets The male and female Buffalo rats used in these experiments were obtained from olonies established here. All animals had free aess to food and water and were maintained at 23'C in a room with lights from 6 to 18 h. Breeding pairs, and pregnant and nursing mothers were fed a standard laboratory how (Prolab mouse, rat and hamster 1 formula; Agway, In., Syrause, NY). Lipid analyses showed that this diet ontained 39.2 mg fatty aid g-' diet of whih 21.2% was linolei aid (Sauer & Dauhy, 1992). Young male and female rats, weighing 3 to 45 g, were separated from their mothers at 21 days of age and were fed ad libitum an essential fatty aid (EFA)-defiient ration (AIN-76 semipurified diet ontaining 5% fat as U.S.P. steari aid, atalog No. 9624, ICN Biohemial, Cleveland, OH). The manufaturer's speifiations indiated that this diet ontained 5% surose, 15% orn starh, 2% asein,.3%
298 L.A. SAUER & R.T. DAUCHY DL-methionine,.2% holine bitartrate, 5% USP steari aid, 3.5% mineral mix and 1% vitamine mix. Lipid analysis (performed in this laboratory) showed that it ontained several long and short hain fatty aids; however, linolei and arahidoni aids were undetetable. Weanling rats fed this diet had a rapid derease in plasma free linolei and arahidoni aid onentrations and an inrease of a new fatty aid, tentatively identified and assumed to be the triene, eiosa-5,8,1 1-trienoi aid, that is formed during EFAdefiieny (Holman, 196). After 8 to 12 weeks on this diet the rats ate about 2-25 g day 1, weighed 225-3 g and were suitable for tumour implantation and as soures of EFA-defiient donor blood. Total plasma free fatty aid onentrations in these animals were.63 ±.24 mm (mean ± s.d., n = 25). Mean onentrations of the six most abundant free fatty aids were: C14:,.1 ±.1 mm; C16:,.2 ±.8 mm; C16:1,.1 ±.5 mm; C18:,.4.1 mm; C18:1,.24 ±.11 mm; and C2:3 (n9),.3 ±.1 mm. Linolei aid was undetetable and arahidoni aid was.1 ±.1 mm. A C2:3(N-9)/ C2:4(N-6) ratio of greater than.4 is indiative of EFAdefiieny (Holman, 196). Tumour implantation and perfusion All experiments were performed with Morris hepatoma 7288 CTC grown subutaneously as tissue-isolated tumours (Sauer et al., 1982). The proedures for implantation of tissueisolated tumours were as desribed (Sauer & Dauhy, 1992). Tumour growth rates were dereased in EFA-defiient rats and a palpable tumour mass was not evident until after a latent period of about 12 days. Growing tumours were used for perfusion when the weight, estimated by measurements made through the skin (Sauer et al., 1986), was 5 to 6 g. Tissue-isolated hepatomas 7288CTC were perfused for 2 h as desribed (Sauer & Dauhy, 1988; 1992). Initial experiments showed that steady state rates of tumour uptake of exogenous linolei aid were established within 15 min after the start of the perfusion. Arterial blood samples for analysis were olleted from a Y-tube plaed in the arterial blood perfusion line immediately before the tumour and venous samples were olleted from the butterfly atheter draining the tumour vein. Rates of tumour fatty aid supply and uptake were alulated as desribed (Sauer & Dauhy, 1992). Both rates have units of ig or nmol min ' g'l tumour. Twenty min before the end of the perfusion the tumour was labelled with [methyl-3h]thymidine (2 yci g- estimated tumour weight, in saline) injeted diretly into the arterial blood atheter (Sauer & Dauhy, 1988). The 3H-thymidine made one pass through the tumour; uninorporated thymidine appeared in the tumour venous blood 2 min after injetion, reahed a peak at 3 to 4 min and was nearly ompletely eliminated from the tumour in 2 min (data not shown). The tumour was rapidly removed from the animal, weighed, and frozen until analysis. A 2% homogenate was made from the thawed tissue in.9% saline solution. Tumour DNA ontent and the 3H-thymidine inorporation into tumour DNA were measured as desribed (Sauer & Dauhy, 1988). Colletion and preparation of donor blood Eighty to 9 ml of whole blood were olleted from 1 heparinised adult, EFA-defiient rats and filtered through two layers of heeseloth. These whole blood preparations were used in the perfusion experiments to determine the ontrol rates of 3H-thymidine inorporation in the absene of EFAs (the baseline rate of 3H-thymidine inorporation). Idential pooled whole blood samples olleted from donor EFA-defiient rats were used in other experiments to determine the effets of single fatty aids or mixtures of fatty aids. To perform these experiments the donor EFA-defiient blood was supplemented with exogenous fatty aid(s), as follows: the whole blood preparations were separated into ells and plasma by entrifugation. One or two solid, purified fatty aids (as sodium salts) were added to the plasma fration and dissolved by warming to 1-15 C with gentle mixing. An amount of the ellular fration was added bak to the plasma to give a reonstituted whole blood mixture with a hematorit of 5%. For most experiments the onentrations of the exogenous free fatty aids are given as plasma onentrations (mm), sine these were analysed diretly. Beause the plasma was diluted 1:1 by ells, the free fatty aid onentration in the whole blood perfusate was one-half the plasma onentration. To determine that the exogenous free fatty aid remained as a free fatty aid and was bound to albumin, we determined the molar ratios for free fatty aid/albumin in EFAdefiient plasma samples (and isolated albumin and globulin frations) and in plasma samples supplemented with linolei aid. Heparinised blood (45 ml), olleted from 5 EFAdefiient donor rats, was entrifuged to obtain a pooled plasma fration (21 ml). The plasma was dialysed for 48 h in the old against phosphate-buffered (5 mm) saline, ph 7.4, and divided into two 1ml portions. One portion was untreated and 1.4 mg sodium linoleate was dissolved in the other. The treated and untreated plasma samples were separated into albumin and globulin frations by hromatography on Affigel blue (Travis & Pannel, 1973). Samples of the EFA-defiient and linoleate-treated plasmas and the albumin and globulin frations isolated from them were analysed for free fatty aids and albumin (Doumas & Biggs, 1972). The albumin onentration in the plasma samples was.36 mm. The free fatty aid/albumin molar ratios in untreated and sodium linoleate-treated, EFA-defiient plasmas were 2.2 and 4.1, respetively. Similar molar rations were observed in the isolated albumin samples indiating that the added linolei aid was reovered bound to albumin. Globulin frations were free of albumin and ontained no detetable free fatty aids. Analysis of the free linolei aid ontent in treated and untreated plasma samples and separation of the plasma lipids by thin-layer hromatography (Sauer & Dauhy, 1992) showed that all of the exogenous fatty aid was reovered in the free fatty aid fration. No evidene for redistribution of exogenous free fatty aid to another plasma lipid lass was found in the perfusate. Also, no redistribution of the exogenous free fatty aid was found in plasma of venous blood olleted after it had passed through the tumour indiating that the exogenous free fatty aid not taken up by the tumour remained bound to albumin and was not transferred by the tumour to another plasma lipid lass. In some experiments [1-_4C]linolei aid was added to the donor plasma in addition to exogenous unlabeled linolei aid, as desribed (Sauer & Dauhy, 1992). After reonstitution of the whole blood following addition of the ellular fration, these donor blood preparations were used to determine linolei aid uptake and utilisation during the perfusion. Analysis offatty aids Free fatty aids in plasma, isolated albumin or globulin frations were extrated and prepared for analysis as desribed (Sauer & Dauhy, 1988, 1992). Measurements were made using either a Perkin-Elmer Sigma 3 gas hromatograph equipped with a 3.2 mm x 1.8 m 5% diethylene glyol suinate olumn at 2 C (nitrogen as arrier gas) and eletroni integrator (Model 339A; Hewlett-Pakard, Sunnyvale, CA) or a Hewlett-Pakard Model 528A gas hromatograph equipped with a.25 mm x 3 m apillary olumn (Model 233, Supelo In., Bellefonte, PA) at 19 C (helium as arrier gas, split, 1:1) an eletroni integrator (Model 3396A, Hewlett-Pakard) and an autoinjetor (Model 7673A, Hewlett-Pakard). Injetion port and flame ionisation detetor were at 22 C. Fatty aid methyl esters were identified by their retention times ompared to known standards. Statistial analysis Means are presented ± s.d., as indiated, and were ompared by one-way analysis of variane (ANOVA) and the Dunan multiple range test. P <.5 was onsidered signifiant.
PLASMA FREE FATTY ACIDS AND TUMOUR 3H-THYMIDINE INCORPORATION 299 Results Uptake of [J-4C]linolei aid by hepatoma 7288CTC perfused in situ Table I shows the results of experiments in whih two hepatomas 7288CTC were perfused in situ with donor blood from EFA-defiient rats that ontained two different onentrations of '4C-linolei aid. Uptakes of linolei aid mass and radioativity were idential for the two tumours; exept for the first time points, the speifi ativities of '4C-linolei aid in the arterial blood perfusate and the tumour venous blood effluent were nearly idential. Also, the rates of linolei aid supply and uptake were reasonably onstant during the 1 and 2 h of perfusion. '4CO2 was released into the tumour venous blood indiating that the '4C-linolei aid removed from the arterial blood was utilised. The rates of oxidation of 14Clinoleate in experiments 1 and 2 gradually inreased throughout the perfusion. After 6 and 12 min of perfusion, the '4C2 prodution rates by the 2 hepatomas were about 2 and 3.5% of the total 14C-linolei aid uptake, respetively, values that are similar to those observed during '4C-palmiti aid uptake and oxidation in hepatoma 7288CTC perfused in situ (Sauer & Dauhy, 1992). Supply and uptake rates for radioativity (d.p.m. min-' g-' tumour) are not shown in Table I. However, these rates may be alulated by multiplying the rate of '4C-linolei aid uptake (fg min-' g- ) by the speifi ativity (d.p.m. Lgg 1) in the blood sample. The total tumour '4C-linolei aid uptake during the 1 h perfusion in experiment 1 was estimated to be 74.4 1g g-' and 49,98 d.p.m. g' tumour. Total tumour linolei aid ontent at the time of harvest was 922fLgg-' and ontained 64,62 d.p.m. g-1; thus, the '4C-ontent found in the tumour was 13% of the estimated total uptake. In experiment 2, in whih the perfusion was for 12 min, linolei aid uptake was estimated to be 276 gg-' and 12568 d.p.m. g '. Total tumour linolei aid ontent was 721 jig g-' and ontained 12288 d.p.m. g-'; therefore, in experiment 2 the '4C-ontent at time of tumour harvest was 98% of the expeted uptake. Comparison of omega-6 and omega-3 fatty aids Several exogenous omega-6 and omega-3 free fatty aids, adjusted to plasma onentrations of about.5 mm in the arterial blood perfusate, were perfused through tissue-isolated hepatomas 7288CTC to test for their ability to affet tumour 3H-thymidine inorporation. As shown in Table II, lear differenes were noted. Eah of the four omega-6 fatty aids inreased the amount of tumour 3H-thymidine inorporation about 2 to 3.5 times above the mean baseline value observed when untreated EFA-defiient donor blood was the perfusate. Linolei aid was the most effetive omega-6 free fatty aid; gamma-linoleni, dihomo-gamma-linoleni and arahidoni aids had about one-third the effet of linoleate. Neither of the four omega-3 fatty aids affeted tumour 3H-thymidine inorporation. Dose-response relationships Sine linolei and arahidoni aids are the most abundant free omega-6 fatty aids in the arterial blood of rats fed a normal diet and both are taken up by hepatoma 7288CTC in proportion to the rate of supply (Sauer & Dauhy, 1992), we investigated the effet of different onentrations of these fatty aids on tumour 3H-thymidine inorporation. These data are shown in Figure la. The initial plasma onentration used (.9 mm) had no effet; however, perfusion of tumours with inreased onentrations of both fatty aids inreased tumour 3H-thymidine inorporation. Hyperbolishaped dose-response urves were observed. The Vmax rate of inorporation observed with linolei aid was about 3 to 3.5 times greater than that for arahidoni aid, indiating that the differene between these two fatty aids noted in Table II was not simply a onentration effet, but rather, that mole for mole linolei aid was more effetive than arahidoni aid. Despite the higher rate of 3H-thymidine inorporation, tumour DNA ontent was unhanged during perfusion for 2 h with blood ontaining either linolei or arahidoni aids. DNA ontent of ontrol tumours perfused for 2 h with blood from EFA-defiient donor rats was 2.76 ±.7 mg g-' tumour. After 2 h of perfusion with blood supplemented with.7 mm linolei aid or.93 mm arahidoni aid, the tumour DNA ontents were 2.97 ±.9 and 2.76 ±.11 mg g-' tumour, respetively. Mean DNA ontent of all tumours was 2.84 ±.8 mg g-' tumour (n = 54). Figure lb shows the relationships between linolei aid supply and uptake for the tumours perfused in the experiments shown in Figure la. Uptake of linolei aid by hepatomas 7288CTC perfused in situ showed the same diret dependeny on supply that was Table I Supply and uptake of arterial blood free [1-'4C]-linolei aid in hepatoma 7288CTC perfused in situ [1-4C]-Linolei aid Speifi Perfusion 14C2 Content Content ativity Supply Uptake Content Release time Tissue fg ml- d.p.m. ml-' d.p.m. dg' lag min-'g- ltg min-' g- d.p.m. ml-' dp.m. ming- Experiment 1 15 min A 17 589 55 2.28 142 V 38 2859 758 1.52 223 2 3 min A 98 6357 65 2.9 139 V 46 2986 648 1.17 522 8 45 min A 95 5951 641 2.3 14 V 53 341 581.97 82 13 6 min A 17 6265 587 2.28 121 V 5 3323 671 1.28 867 15 T 922a 6462b 7 Experiment 2 3 min A 178 7571 425 4.7 V 67 352 525 2.65 75 16 6 min A 165 7914 479 4.4 176 V 72 3552 521 2.51 145 27 12 min A 148 869 545 3.63 185 V 76 4534 597 2.2 1813 35 T 72 la 12288b 17 Experiments I and 2: A = arterial blood; V = tumour venous blood; T = tumour; aslg g- I; blad.p.m. g I; and assumes that all radioativity in the tumour was 14C-linonei aid. In experiment 1: total tumour '4C-linoleate uptake was 4998 d.p.m. g-'; tumour weight was 6.23 g; arterial and venous blood flow were.133 and.124mlmin-', respetively; and the linoleate onentration in arterial blood was.36mm. In experiment 2: total tumour '4C-linoleate uptake was 125,68 d.p.m. g '; tumour weight was 6.12 g; arterial and venous blood flow were.15 and.13mlmin-'; and the linoleate onentration in arterial blood was.58mm.
3 L.A. SAUER & R.T. DAUCHY 4.2 3 o.z < Z L-a -.C 1a 2.E._aE Table II Effets of omega-6 and omega-3 fatty aids on 3H-thymidine inorporation in hepatoma 7288CTC perfused in situ Tumour Plasma Tumour fatty 3H-thymidine onentration aid uptake inorporation Fatty aid added mm nmolg-'min-' d.p.m. l.g- DNA None 44 6 Linolei aid.5 1.8 ±.9 342 ± 8b, (18:2n6) y-linoleni aid.52 3.7 ± 1. 147 ± 15b (18:3n6) Dihomo-y-Linoleni aid.67 8.1 ±.8 145 ± gb (2: 3n6) Arahidoni aid.46 2.9 ±.6 122 ± 12b (2:4n6) a-linoleni aid.71 1.9 ± 1. 45 ± 5a (18:3n3) Otadeatetraenoi aid.56 1.6 ±.6 4 ± 4a (18:4n3) Eiosapentaenoi aid.43 2.4 ±.9 5± 3a (2: 5n3) Doosahexaenoi aid.54 1.6 ±.4 43 ± 3a (22:6n3) athese values are not different (P >.5) from values obtained in the absene of added fatty aid. bthese values are different (P <.1) from values obtained in the absene of added fatty aid or in the presene of the omega-3 fatty aids. Values for linolei aid are different (P <.1) from those for other omega-6 fatty aids. Tissue-isolated tumours weighing 5-6 g were perfused for 2 h in situ with whole blood olleted from essential fatty aid-defiient rats. Exogenous fatty aid was added, as indiated. Values are means ± SD for three experiments. a observed in in vivo tumours growing in EFA-suffiient rats (Sauer & Dauhy, 1992). Most importantly, the uptake of linoleate was dependent on supply throughout the plasma onentrations examined. Therefore, the stimulative effet of plasma free linolei aid on tumour 3H-thymidine inorporation reahed a plateau while the rate of linolei aid uptake was still inreasing; the linolei aid dependent reation was saturated. C 1 I EC E C. o..2.4.6.8 1. Plasma fatty aid onentration, mm 1 8 6 4 2 7.. *7/ u n 2 4 6 8 1 12 Supply, nmol min-1 g-1 Figure la The relationship between the arterial plasma linolei or arahidoni aid onentration and 3H-thymidine inorporation into hepatoma 7288CTC DNA; () linolei aid; () arahidoni aid. Eah point represents the mean ± s.d. for perfusions performed on three different tumours. b, The relationship between the supply and uptake of free linolei aid by hepatoma 7288CTC. These data are from the same perfusions shown in a. Results of the regression analysis were: slope =.696, interept =.48, and orrelation oeffiient =.9496.. * b Competition among omega-6, omega-9 and omega-3 fatty aids Interations among olei, arahidoni, alpha-linoleni or eiosapentaenoi aid and linolei aid were examined by ombining the free fatty aids in the arterial blood perfusate and measuring the 3H-thymidine inorporation in tumours perfused with the mixtures. As shown in Table III, olei aid, at a high plasma onentration, did not alter the stimulative effet of linolei aid. (We have previously shown that saturated fatty aids or olei aid had no effet on tumour 3H-thymidine inorporation when added alone (Sauer & Dauhy, 1988)). The response due to arahidoni aid appeared to be additive to that of linoleate and both alphalinoleni and eiosapentaenoi aids (at onentrations about equal to that of linoleate) inhibited linolei aid uptake and the stimulative effet on 3H-thymidine inorporation by about two-thirds. The inhibitory effet of these omega-3 fatty aids was examined further by perfusing individual tumours with arterial whole blood ontaining a fixed plasma onentration of linolei aid (.5 mm) and different plasma onentrations of either alpha-linoleni or eiosapentaenoi aid that ranged from to about.9 mm (Figures 2a and b). Inreasing onentrations of these omega-3 fatty aids inhibited both tumour linolei aid uptake and the positive effet of linoleate on 3H-thymidine inorporation. Dixon plots of these data (shown in the insets) indiate that the inhibitory effet of alpha-linoleni and eiosapentaenoi aids on both funtions was ompetitive. Ki values for alpha-linoleni aid were.18 mm for the inhibition of linoleate uptake and.25 mm for the inhibition of 3H-thymidine inorporation. The uptake of the other endogenous fatty aids in the perfusate was also inhibited by these two omega-3 fatty aids (data not shown).
PLASMA FREE FATTY ACIDS AND TUMOUR 3H-THYMIDINE INCORPORATION 31 Table II Effets of olei, arahidoni, a-linoleni and eiosapentaenoi aids on linolei aid uptake and the stimulation of 3H-thymidine inorporation by linolei aid in hepatoma 7288CTC perfused in situ Tumour Plasma Tumour fatty 3H-thymidine onentration aid uptake inorporation Fatty aids added mm nmolg 'min-' dp.m. lg' DNA Linolei aid Arahidoni aid.5.47 1.8.9 3.3 ±.7 342± 8 122 ± 12 Linolei aid.64 11.1 ±.9 Olei aid.99 1.9 ±.6 361±2 Linolei aid.47 5.8 ±.6 Arahidoni aid.47 8.3 ± 1.3 459± 16a Linolei aid.57 2.6 ±.9 a-linoleni aid.59.4.2 127 7a Linolei aid.52 2.3 ±.2 Eiosapentaenoi aid.36 2.7 ±.4 135±6a Values are means ± SD for three experiments. athese values are different (P <.5) from the mean value obtained in the presene of linolei aid alone. a b C 4- )I_._ 3._E Ci 12 ) 1.2 a'i -a7.8.4.8 4Conentration, mm. o ) CB Q I S z a ) E - 1.2 Plasma omega-3 fatty aid onentration, mm Figure 2 Effets of alpha-linoleni and eiosapentaenoi aids on the rates of linolei aid utilisation a, and linolei aid-dependent 3H-thymidine inorporation b, by hepatoma 7288CTC. Hepatomas 7288CTC were perfused for 2 h in situ with donor blood ontaining added linolei aid (.5 mm) and,.5,.12,.28,.59, or.94 mm alpha-linoleni () aid or,.1 or.36 mm eiosapentaenoi () aid. Eah point represents mean ± s.d. for perfusions performed on three different tumours. The insets are plots of reiproal rates against the omega-3 fatty aid onentrations. Disussion The purpose of this study was to determine the effets of omega-6 and omega-3 free fatty aids on 3H-thymidine inorporation in tissue-isolated hepatomas 7288CTC perfused in situ. This tumour model repliates the in vivo ondition and removes the tumour from host effets that annot be ontrolled; we hoped these experiments would aid understanding of how these fatty aids affet tumour growth. Choies were made in the seletion of the nutritional state of the animals and in methods of preparing the whole blood perfusate and should be disussed. EFA-defiient animals were used for tumour growth and as soures of donor blood. In previous experiments (Sauer & Dauhy, 1988), we used rats fed normal laboratory how for these purposes. The tumours ontained large amounts of linolei and arahidoni aids and the linolei and arahidoni aid onentrations in donor blood plasma ranged from less than.1 to greater than.3 mm, depending on the feeding ativity of the host rats (Sauer & Dauhy, 1992). Baseline rates of 3H-thymidine inorporation in tumours perfused with this blood were about 1 d.p.m. Lg-' DNA and were inreased to about 16 d.p.m. pg-' DNA by.7 mm plasma linolei aid. It seemed likely that these baseline rates ould be further dereased if the tumours were grown in EFA-defiient rats and were perfused with donor blood from other EFA-defiient rats. The response of the tumour to exogenous linoleate might also be inreased. These expetations were observed; baseline levels of 3H-thymidine inorporation were dereased to about 4 d.p.m. sg-' DNA and the Vmax response of the perfused tumour to.7 mm plasma linoleate was inreased to about 35 d.p.m..tg-' DNA. Clearly, EFA-defiieny influenes the metabolism of both the host and tumour: host animals were very suseptible to water loss and dehydration and tumours implanted in these animals grow more slowly (Hillyard & Abraham, 1979; Sauer & Dauhy, 199). Despite the slower tumour growth, the important reations in this study, fatty aid uptake and utilisation and tumour 3H-thymidine inorporation, remained intat. Most important were the qualitatively idential responses in 3H-thymidine inorporation of tumours growing in EFA-suffiient rats (Sauer & Dauhy, 1988) and EFAdefiient rats (Table III and Figure 1) during perfusion with inreased onentrations of linolei or arahidoni aids. The larger, more signifiant level of 3H-thymidine inorporation was observed in tumours growing in EFA-defiient rats. Beause tumours grown in EFA-defiient rats have low endogenous levels of essential fatty aids and respond briskly to exogenous linolei aid, we believe this host-tumour model
32 L.A. SAUER & R.T. DAUCHY will be useful in experiments designed to determine mehanisms of ation. It is important to note that the EFA-defiient rats were fed a diet that ontained 2% protein,.3% methionine, 65% arbohydrate, and 5% fat, plus vitamins, minerals, and holine bitartrate equivalent to that in normal rat how. Blood FFA and lipoproteins from these animals ontained saturated, monounsaturated and eiosa-5,8,11-trienoi aids. Only the essential fatty aids and their metabolites were low or absent. Body growth was slower than that of animals fed an EFA-suffiient diet, and it ontinued throughout the life of the rats. Also, EFA-defiient animals aumulated fat stores indiating that the energy supply was adequate. Pooled donor blood removed from EFA-defiient rats (used for perfusion) was idential in nutrient ontent to blood in the host rat and had no effet on baseline tumour 3H-thymidine inorporation. Rather, the stimulative effet was observed only in tumours perfused with donor blood ontaining linolei, gamma-linoleni, dihomo-gamma-linoleni or arahidoni aid. It seems very unlikely, therefore, that the stimulative effets on tumour 3H-thymidine inorporation illustrated in Figure la were aused by an endogenous fator in the pooled blood from EFA-defiient rats. In agreement with this reasoning, we showed that tumour growth in EFA-defiient rats was speifially inreased by exogenous linolei aid (Sauer & Dauhy, 199). FFA metabolism in perfused organs, suh as liver, is most often studied using perfusates formed by adding a FFAbovine serum albumin omplex to whole blood or to buffered-erythroyte suspensions (Nestel & Steinberg, 1963; Van Harken et al., 1969; Soler-Argilaga et al., 1974). Addition of FFA-albumin omplexes dilutes the original plasma and may add unwanted proteins and peptides to the perfusate. Although there was no evidene that FFA uptake from a rat plasma-bovine albumin-ffa mixture would not our or would be different from normal rat plasma, we deided to avoid the effet of dilution and the presene of foreign proteins. Initial attempts to add FFA diretly to rat plasma using a solid resin proedure (Spetor & Hoak, 1969) were not ompletely suessful beause, in our hands, FFA transfer from elite to albumin was variable and the plasma FFA onentration needed to be measured before the perfusion was performed. The perfusates used in these experiments were prepared by adding purified FFAs (as sodium salts) diretly to the donor blood plasma. The ellular fration was then added bak to reonstitute the whole blood. This proedure was quik, the transfer of FFA to rat plasma albumin was stoihiometri, no other potentially harmful proteins or peptides were added, and the FFA did not redistribute to other plasma lipids in either the arterial blood perfusate or the tumour venous blood. Most importantly, tumour supply and uptake of the exogenous FFA were not detetably different from that of endogenous FFAs. The mehanisms by whih plasma free linolei and arahidoni aids inrease 3H-thymidine inorporation in tumours perfused in situ have yet to be determined. The data reported here suggest that these fatty aids may at differently. Mole for mole, linolei aid onsistently had a greater stimulative effet than did arahidoni aid. This differene is seen in the dose-response relationships illustrated in Figure 1. The Vmax for tumour 3H-thymidine inorporation during perfusion with linoleate was about three times greater than that with arahidonate. Also, the reations due to eah fatty aid were saturable. Experiments in whih linolei and arahidoni aids were ombined in the perfusate (Table III, and see Sauer & Dauhy, 1988) suggested that the effets of the two aids were additive. Therefore, it seems very unlikely that linoleate and arahidonate at through a single, ommon reation. Both linolei and arahidoni aids are substrates for further enzyme oxidations in ells. Coneivably, these fatty aids (or their metabolites) ating independently of eah other inrease the rate of DNA synthesis of tumour ells in ative S phase. Alternatively, these fatty aids ould at to reruit new ells into S phase and/or to ativate ells arrested in S phase. Eah of these ations would be measured as an inrease in of 3H-thymidine inorporation and ultimately as an the rate inrease in tumour DNA ontent. However, during the 2 h perfusion no inrease in tumour DNA ontent was observed, even in those tumours showing the highest rates of 3Hthymidine inorporation (see also Sauer & Dauhy, 1988). Presumably, not enough new DNA was formed to be deteted hemially. Sine perfusions longer than 2 h are diffiult to omplete suessfully, the question of amounts of new DNA synthesised must be examined using more sensitive analytial methods. Finally, a plausible and non-trivial explanation would result if either linoleate or arahidonate (or both) ated to derease the thymidine pool size in the tumour ells. Suh a hange ould inrease the effetive speifi ativity of the administered 3H-thymidine dose and inrease the amount of 3H-thymidine inorporated without hanging the tumour DNA ontent. In our opinion, mehanisms based on hanges in intraellular thymidine pools are the least satisfatory beause (1) two different thymidine pool size regulatory reations would appear to be required to explain the different stimulative effets of linolei and arahidoni aids (Figure 1) and (2) the dereases in pool sizes would need to be additive (Table III). Also, it is diffiult to envisage how dereases in a thymidine pool ould be responsible for the inreased tumour growth and DNA ontent assoiated with longer exposures to high plasma free linolei and arahidoni aid onentrations (Sauer et al., 1986; Sauer & Dauhy, 1987). Unfortunately, analyses of preursor pools are diffiult to interpret espeially in solid tumours omposed of several different ell types; determination of an average nuleotide pool has no biohemial meaning in a solid tumour. We believe the simplest, most testable explanation for these data is that ambient onentrations of plasma free linolei and arahidoni aids at positively via onentration dependent reations to inrease the rate of DNA synthesis in tumours ells that are in S phase. Clearly, these early events in linoleate- and arahidonate-stimulated tumour 3H-thymidine inorporation are imperfetly understood and require further experimentation. It is of interest to ompare the speifiity of fatty aid requirements in: tumour growth in vivo (see Welsh, 1987 for a review); 3H-thymidine inorporation in tumours perfused in situ; and 3H-thymidine inorporation and growth in aner ells in ulture. Similarities and differenes disovered using these three different systems are pertinent. Despite early reports that high fat diets inreased tumour growth, evidene now points to dietary linolei aid intake as the ritial fator (Hillyard & Abraham, 1979; Ip et al., 1985). Ingested arahidoni aid had no effet (Hillyard & Abraham, 1979). In solid tumours perfused in situ, linolei aid aused a large response in 3H-thymidine inorporation; arahidoni, gamma-linoleni and dihomo-gamma linoleni aids were also ative, albeit at about one-third the ativity of linolei aid (Sauer & Dauhy, 1988; and see Figure 1 and Table II). Perfusion with saturated or monounsaturated fatty aids had no effet (Sauer & Dauhy, 1988). Rodent and human tumour ells in ulture show inreased rates of ell proliferation (Holley et al., 1974; Wiha et al., 1979; Rose & Connelly, 199) and inreased rates of 3H-thymidine inorporation (Wiha et al., 1979; Rose & Connelly, 199) when linolei, arahidoni or olei aid was inluded in the medium. Thus, the fatty aid requirement for growth of tumours in vivo, for 3H-thymidine inorporation in tissue-isolated perfused tumours, or for 3H-thymidine inorporation and growth of tumour ells in ulture is haraterised by a progressive derease in fatty aid speifiity. Studies of mehanism require models that dupliate the intat animal; we believe that the tissue-isolated tumour perfused in situ serves this purpose best. The mehanism of ation of the omega-3 fatty aids is also not known. Ingestion of these fatty aids has an inhibitory effet on tumour growth in vivo (Karmali et al., 1984; Gabor & Abraham, 1986) and, when omega-3 fatty aids were ingested with linolei aid, the positive growth effets of linolei aid were dereased (Gabor & Abraham, 1986). Experiments reported here show that alpha-linoleni and
PLASMA FREE FATTY ACIDS AND TUMOUR 3H-THYMIDINE INCORPORATION 33 eiosapentaenoi aids inhibited both linolei aid uptake and the stimulative effet of linoleate on tumour 3H-thymidine inorporation (Table III and Figure 2). Assuming that inhibition of 3H-thymidine inorporation in a tumour perfused in situ and inhibition of growth of a tumour mass in the intat rat are the first and the final stages of the same inhibitory proess, one ould expet similar mehanisms. In support of this reasoning, it is known that feeding diets ontaining omega-3 fatty aids either alone or in ombination with linolei aid aused a substantial derease in the tumour ontent of linolei aid (Gabor & Abraham, 1986), suggesting a derease in uptake. It seems likely, therefore, that ingestion of dietary oils ontaining omega-3 fatty aids inreased the ambient levels of plasma free omega-3 fatty aids in host arterial blood and this inrease ompetively inhibited tumour linoleate uptake. Sine linolei aid uptake appears to be ritial for determining the tumour growth rate, the result is an omega-3 fatty aid-mediated derease in tumour growth. Further experiments designed to test this hypothesis are in progress. This researh was supported by Grant No. CA2789-11 from the National Caner Institute, NIH and Grant No. 9A42 from the Amerian Institute for Caner Researh. We wish to thank Dr Estelle Goodell and Heidi Johnson for help in preparing the Tables. Referenes ABRAHAM, S. & HILLYARD, L.A. (1983). Effets of dietary 18- arbon fatty aids on growth of transplantable mammary tumors in mie. J. Natl Caner Inst., 71, 61-65. DOUMAS, B.T. & BIGGS, H.G. (1972). Determination of serum albumins. In Standard Methods in Clinial Chemistry, Cooper, G.A. (ed) p. 175-188, Vol. 7, Aademi Press: New York. GABOR, H., HILLYARD, L.A. & ABRAHAM, S. (1985). Effet of dietary fat on growth kinetis of transplantable mammary arinoma in BALB/ mie. J. Natl Caner Inst., 74, 1299-135. GABOR, H. & ABRAHAM, S. (1986). Effet of dietary menhaden oil on tumour ell loss and the aumulation of mass of a transplantable mammary adenoarinoma in BALB/ mie. J. Natl Caner Inst., 76, 1223-1229. HILLYARD, L.A. & ABRAHAMS, S. (1979). Effet of dietary polyunsaturated fatty aids on growth of mammary adenoarinomas in mie and rats. Caner Res., 39, 443-4437. HILLYARD, L.A., RAO, G.A. & ABRAHAMS, S. (198). Effet of dietary fat on fatty aid omposition of mouse and rat mammary adenoarinomas. Pro. So. Exp. Biol. Med., 163, 376-383. HOLLEY, R.W., BALDWIN, J.H. & KIERNAN, J.A. (1974). Control of growth of a tumour ell by linolei aid. Pro. Natl Aad. Si. USA, 71, 3976-3978. HOLMAN, R.T. (196). The ratio of trienoi:tetraenoi aids in tissue lipids as a measure of essential fatty aid requirement. J. Nutrit., 7, 45-41. IP, C., CARTER, C.A. & IP, M.M. (1985). Requirement of essential fatty aid for mammary tumorigenesis in the rat. Caner Res., 45, 1997-21. JURKOWSKI, J.J. & CAVE, W.T. (1985). Dietary effets of menhaden oil in the growth and membrane lipid omposition of rat mammary tumors. J. Natl Caner Inst., 74, 1145-115. KARMALI, R.A., MARSH, J. & FUCHS, C. (1984). Effet of omega-3 fatty aids on growth of a rat mammary tumour. J. Natl Caner Instit., 73, 457-461. NESTEL, P.J. & STEINBERG, D. (1963). Fate of palmitate and of linoleate perfused through the isolated rat liver at high onentrations. J. Lipid Res., 4, 461-469. ROGERS, A.E. & WETSEL, W.C. (1981). Mammary arinogenesis in rats fed different amounts and types of fat. Caner Res., 41, 3735-3737. ROSE, D.P. & CONNOLLY, J.M. (199). Effets of fatty aids and inhibitors of eiosanoid synthesis on the growth of a human breast aner ell line in ulture. Caner Res., 5, 7139-7144. SAUER, L.A., STAYMAN, J.W. & DAUCHY, R.T. (1982). Amino aid, gluose, and lati aid utilization in vivo by rat tumours. Caner Res., 42, 49-497. SAUER, L.A., NAGEL, W.O., DAUCHY, R.T., MICELI, L.A. & AUSTIN, J. (1986). Stimulation of tumour growth in adult rats in vivo during an aute fast. Caner Res., 46, 3469-3475. SAUER, L.A. & DAUCHY, R.T. (1988). Identifiation of linolei and arahidoni aids as the fators in hyperlipemi blood that inrease 3H-thymidine inorporation in hepatoma 7288CTC perfused in situ. Caner Res., 48, 316-3111. SAUER, L.A. & DAUCHY, R.T. (19). Tumour-host metaboli in'terrelationships. Biohem. So. Trans., 18, 8-82. SAUER, L.A. & DAUCHY, R.T. (1992). Uptake of plasma lipids by tissue-isolated hepatomas 7288CTC and 7777 in vivo. Br. J. Caner, 66, 29-296. SOLER-ARGILAGA, C., INFANTE, R., RENAUD, G. & POLONOVSKI, J. (1974). Fators influening free fatty aid uptake by the isolated perfused rat liver. Biohimie, 56, 757-761. SPECTOR, A.A. & HOAK, J.C. (1969). An improved method for the addition of long-hain free fatty aids to protein solutions. Anal. Biohem., 32, 297-32. TRAVIS, J. & PANNEL, R. (1973). Seletive removal of albumin from plasma by affinity hromatography. Clin. Chem. Ata., 49, 49-52. VAN HARKEN, D.R., DIXON, C.W. & HEIMBERG, M. (1969). Hepati lipid metabolism in experimental diabetes. V. The effet of onentration of oleate on metabolism of triglyerides and on ketogenesis. J. Biol. Chem., 244, 2278-2285. WELSCH, C.W. (1987). Enhanement of mammary tumorigenesis by dietary fat: review of potential mehanisms. Am. J. Clin. Nutr., 45, 192-22. WICHA, M.S., LIOTTA, L.A. & KIDWELL, W.R. (1979). Effets of free fatty aids on the growth of normal and neoplasti rat mammary epithelial ells. Caner Res., 39, 426-435.