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JACC Vol. IP, No .7 1824 December 1991 :1R24-8
SEMINAR ON CARDIOVASCULAR MANIFESTATIONS OF THE TOXIC OIL SYNDROME AND RELATED CONDITIONS-V* Thomas N . James, MD, FACC, Guest Editor
Synergistic Vascular Toxicity and Fatty Acid Anilides in the Toxic Oil Syndrome
PAUL J . BOOR, MD . M . FIROZE KHAN, PHD, BHUPENDRA S . KAPHALIA, PHD, THOMAS R . JERRELLS . PHD, G . A. S . ANSARI, PHD Galve .vton, Texas
The underlying etiology of the toxic oil syndrome may be related alterations during cooking is supported by high pressure liquid to any of several toxic contaminants . The hypothesis is made that chromatographic analysis. two or more toxic compounds may act synergistically to cause The theoretic metabolism of fatty acid anilides is discussed. vascular damage in the toxic oil syndrome. To support this Recent data concerning the toxic actions of the anilides of oleic and hypothesis, previous studies are reviewed concerning the remark. linoleic acid are given . These data suggest that these anllides able synergistic toxic action of allylamine and beta-aminopropi. induce immunologic alterations that may be similar to those seen onitrile on the media of blood vessels . Although these toxins are in the toxic al syndrome. In addition, the heated anilidas appear not directly related to the toxic oil syndrome, this previous to have increased toxicity, supporting the concept that the use of experimental work emphasizes the possibility that unexplored toxic oil in cooking may Increase Its toxicity . synergistic actions may be important. Furthermore, the hypothe- (J Am Coil Cordial 199) ;18:1824-8) sis that contaminating fatty acid collides in toxic oil undergo
The toxic oil syndrome associated with the use of adulter- Our laboratory (17) has been interested in vascular toxins ated rapeseed oil in humans has been well described (1-11), for several years, especially in synergistic toxic interactions although the precise underlying mechanisms of toxic injury between environmental compounds that may adversely af- to a variety of tissues remains enigmatic . This problem is fect blood vessels . In our initial experimental approach to compounded by the lack of a clear-cut experimental model the toxic oil syndrome, we hypothesized that some alteration that displays the entire array of clinical findings and patho- occurred during cooking in the contaminated oil, resulting in logic lesions found in humans, despite experimental studies enhanced toxic effects . We focused on the fatty acid anilides of toxic oil in several species (12-16) . Although vascular as potential candidates for the active toxins in toxic oil lesions have been proposed to play a major role in the syndrome and specifically addressed their toxicity and heat- vasculotoxicity has not been clinical syndrome, definite induced structural modifications that might result in en- clearly demonstrated in experimental animals . hanced toxic effects. In this report we first review our background work with From the Department of Pathology. University cf Texas Medical Brunch, two specific vascular toxins: allylamine and beta-aminopro- Galveston, Texas . This work was mpponed by Grant HL-26189 and ES-04815 pionitrile . Although not directly related to the toxic oil from the National Heart, Lung, and Blood Institute, National Institutes of syndrome, this experimental work emphasizes the possibil- Health. Bethesda, Maryland and the Institmo National De La Salad . Spain through the World Health Organization. Regional Office for Europe, Copes' ity that unexplored synergistic interactions may play a role. hagen. Denmark . Then we present our recent data with oleic and linoleic acid Manuscript received April 15. 1991 ; accepted June 17, 1991 . Address far in, Paul 1 . Boor . vIa. University of Teas. Medical anilides, which indicate that their systemic toxic effects are Branch. Department of Pathology . 232 Keiller Building. F-05 . Gal-ton. enhanced by the formation of several distal unknown com- Texas 7755(r pounds during heating and support the concept that the toxic 'Pans I to IV of this Seminar appeared in the September, October. November I and November 15 issues of the Journal O Am Coll Cordial effects found in the toxic oil syndrome are related to the use 1991 :18:707-17;1043-7 ; 1367-9, 1539-45) . of adulterated rapeseed oil in cooking.
01991 by the American College of Cardiology 0735-1097191153.50
JACC Vol, 19. No . 7 BOOR ET AL . 1825 December 1991:1024-a SYNERGISTIC VASE CLAR TOXINS IN TOXIC OIL SYNDROME
Figure 1. Synergistic toxicity of vascular toxins . Photomicrographs of thoracic aorta from rats treated with allylamine 1100 mglkg) or beta-aminopropionitrile 11 glkg). or both, A, In rats treated for Ia days with only one of these toxins, the micrographs show normal stmcture with layers of elastic fibers inteLpeeved with vascular smooth muscle cells . The sur- face of the lumen is to the right and the adventitia is m the left in all micrographs . B, In rats given both toxins, massive smooth muscle cell necrosis of the mid- media (m) occurs. C, Nods are allowed to survive for 3 to 6 months after treatment with both toxins, medial necrosis persists and the s::herdothelial layer undergoes cartilaginous metaplasia (arrows). A to C. Hematoxytin-eosin x300. reduced by 30% .
Synergistic Vascular Toxicity toxic oil syndrome could be the toxic agents responsible Combination of vascular toxins- We recently described a for the clinical syndrome. Because oleic acid anilide and linoleic acid anilide constituted 64% and 24%o, respectively, synergistic toxic effect (17) of allylamine, an unsaturated primary amine with minor industrial usage that has been of the total fatly acid anilides in toxic oil samples (30), we known to be cardiotoxic for many years (18-22) . and beta- studied the toxicologic responses of rats to purified oleic aminopropionitrile, another well studied toxin that causes acid anilide and linoleic acid anilide. In addition, we also aortic aneurysm when given for prolonged periods of time to studied the responses of rats to heated oleic acid anilide experimental animals (23-26) . In these experiments- groups (300'C for 30 mini and heated linoleic acid anilide (200°C of male Sprague-Dawley rats (160 to 230 g) were given by for 30 min) with the presumption that these fatty acid Savage either allylamine (100 mg/kg body weight) alone . anilides might be oxidized during cooking, and oxidized beta-aminopropionitrile (1 glkg) alone or both chemicals at compounds or their secondary decomposition products these doses . Rats were Savaged daily for a total of 10 days might play a critical role in the pathogenesis of the toxic oil and studied by standard histochemical methods from I to 10 syndrome. days. Lesions in the aorta, myocardium and muscular arter- Hypothetical metabolites of fatty acid anilides. The hypo- ies of the tongue were assessed . thetical effects of heating (or in vivo metabolism) of oleic Arterial pathology (Fig . 1) . A striking synergistic toxicity acid anilide are summarized in Figure 2. The effect of heating was observed in rats treated with both compounds compared in the presence of ambient air (or metabolism through P-450 with groups of rats given either compound alone, Rats given or lipoxygenase) could lead to the formation of an eposide allylamine plus beta-aminopropionitrile developed massive that was either degraded at this point or further metabolized smooth muscle cell necrosis of the mid-media of the aorta . to a p-hydroxy derivative by the actions of an enzyme such characterized by extensive vacuolation of cells and frank as aniline hydruxylase, which on further oxidation would necrosis with loss of nuclear and cytoplasmic detail. In lead to the formation of immunoquinone . All of these com- addition, medium-sized muscular arteries in the tongue pounds have the capacity for covalent binding and can showed peculiar cytoplasmic inclusions in vascular smooth further decompose to products of unknown nature . The muscle cells after combined treatment ; these eosinophilic same types of compounds could also be formed by an globules were most prominent in the combined treatment ah:rnate route, whereby hydroxylation of aniline leads to group, but occurred to a lesser extent in rats given allylamine the formation of a p-hydroxy derivative and a new com- only . Subsequent exposure of cultured porcine aortic pound . N-acetyl immunoquinone has been identified as a smooth muscle cells as previously described (27,28) also toxic metabolite of aeetamtnnphen and shown to be involved demonstrated a synergistic effect of allylamine plus beta- in covalent binding with macromolecules (31,32) . The ep- aminupropionitrile on cultured medial cell viability in vitro . oxide of linoleic acid anilide has also been found in oil samples associated with ;he toxic oil syndrome (33). A third possibility is the peroxidation of oleic acid anilide, which Oleic Acid and Linoleic Acid Anilides in the may result in the formation of compounds containing hy- Toxic Oil Syndrome droperoxy. hydroxy, keto and aldehydic groups that may be Several investigators 111,12 29) have suggested that fatty imclved in covalent binding to macromolecules . 4Hydroxy- acid anilides found in the cooking oil associated with the eonenol, and alpha- aad beta-unsaturated aldehyde resulting
. No 1 82 6 BOOR ET AL . JACC Val . IS . 7 SYNERGISTIC VASCULAR TOXINS IN TOXIC OIL SYNDROME December 1991 :1824-8
I Figure 2 . Metabolic scheme for the simplest and most abundant oleic acid nailide (1) found in toxic oil samples . The alterations shown may occur through in vivo metabolism or through heating of anilide during cooking con- ditions . Epoxide formation (II) is a likely oxi- dative step, with subsequent metabolism to a p-hydroxy derivative (III) by an enzyme such as aniline hydroxylase. Further oxidation to )mmunoquinone (IV) would be expected . An alternate pathway involves hydroxylation of the aniline to form compound V . Such a scheme would become increasingly complex for anilides containing more double bonds, such as linoleic acid anilide . See text for d: . tails.
° IV
from lipid peroxidation of fatty acids have been reported in fatty acid anilides and their heated counterparts ; these data toxic oil syndrome oil samples (34) . will be summarized here. Male Sprague-Dawley rats (ISO to Oleic acid anilide. This is the major and simplest unsatu- 190 g) were given 250 mg/kg of oleic acid anilide, heated oleic rated anilide found in oil samples responsible for the toxic oil acid anilide and linoleic acid anilide or heated linoleic acid syndrome . The complexity seen with this simplest anilide anilide in mineral oil by gavage on alternate days for 2 weeks will be greatly magnified with the increasing number of (total seven doses) . The control rats received an equal double bonds in anilides such as linoleic, linoleic and ara- volume of mineral oil only . Rats were killed at day 1, 7 or 28 chidonic acid anilides . An example of the analysis of heated after the last dose . Hematologic analyses on day I showed oleic acid anilide by high performance liquid chromatogra- an increase in white blood cell counts in the rats receiving phy is shown in Figure 3 . heated oleic acid anilide and at day 28 in both the oleic acid anilide and the heated oleic acid anilide group, possibly indicating an ongoing subtle inflammatory response, al- Review of Recent Data though no microscopic morphologic lesions were seen in Experiments with purified fatty acid anilides . We recently complete autopsy specimens . The rest of the blood compo- reported (35-37) the results of experiments with purified ncnts measured were not affected . In contrast, rats treated with linoleic acid anilide and heated linoleic acid anilide showed a decrease in red blood cell counts and hemoglobin Figure 3 . Reversed phase high performance liquid chromatographic elation profile of oleic acid anilide (a) and oleic acid anilide heated at content and an increase in platelet counts in the heated 300°C for 30 min to simulate cooking conditions (b). In both cases . linoleic acid anilide group at day I, indicating an effect on the 0.5 gg of compound was injected and eluted with methanol :water hemopoietic system . However, these changes seem to be (7 :1 . vol/vol, flow rate 1 .25 ml/mis . Heated anilide shows additional associated with the concentration of the anilide or its decom- unknown peaks and decreased peak of pure heated oleic acid position products in the biophase because at later time anilide . AUFS = absorption units full scale . points, these measurements returned to control levels . Effects on the immune system. Among the important observations from these investigations were the abnormali- ties in the immune system as a result of exposure to these E anilides . Fatty acid anilides have been reported to be immu- nogenic (12) and cause alterations in the synthesis of immu • b The serum immunoglobulin level and com- 8'a noglobulins (14). position are an expression of the response to challenges and fta infections and could serve as an important tool for the 8 clinical diagnosis of the disease. We observed remarkable increases in the serum IgA levels throughout the exposure period in all treatment groups, especially in the heated oleic 6 8 16 24 32 4b 0 a 16 24 32 40 acid anilide and heated linoleic acid anilide groups, which Relent- Time (Min) Rotonban Tone twin) could he in response to a decrease in the levels of secretory
JACC Vet . 18. No. 7 BOOR ET AL . 182- December 1991 :1824-8 SYNERGISTIC a csCLL\R TOXINS IN TOXIC OIL SYNUROxrE
IgA as suggested by McGee and McMurray (38) . Although Furthermore . both of the anilides that we examined in vivo linoleic acid anilide and heated linoleic acid anilide groups appear to have increased toxicity when heated, supporting showed some alterations in IgM and IgG levels . changes the concept that the use of toxic oil in cooking was partially observed were magnitude and inconsisrem . of tow responsible for its eventual devastating effects in humans . Effects on the spleen . The organ that seemed to be the Further studies of these toxic compounds and especially of most affected target of the anilides and their oxidation synergistic effects that may result from their combination products was the spleen, which war, enlarged during most of with other chemicals should help to define further the stages of fatty acid anilide treatment ; again . the heated underlying mechanisms of the toxic oil syndrome. anilides induced greater changes . An increase in the spleen weight has also been reported (39l in many immunologic disorders and diseases, as well as after chemical exposure . References The increase in spleen weight was associated With alter- I . Toxic Epidemic Study Group. Tonic oil syndrome . Spain Lawsi 1981, ations in the percent splenic T lymphocytes and T cell subsets, whereas B cells were not affected . A decrease in 2. Kilbuurne EtI. Rigau-Niez JG . Heath Cw. et al. Clinical epiderniulogy splenic T cells in the heated oleic acid anilide group was of tusk oil atndrome. N Engi I Med 1983:209:1408-I4. Ricny1R.CabeLIcA .RndeiguezL72ilorI .Neurapaihological iud,e,ar obtained at day I and then a significant increase at day 28 the Iaa:e syndrome re lay e d t o aduheraled rapesee d o0 in Spain . 3- was observed. T helper and T suppressor cell numbers 19s11J&817-,, decreased at day I in oleic acid anilide and heated oleic acid 4 . Coolm Gurcia M . Pwada de la Paz IM. Dim de noon F. I W. Hypcrco- aeulable -tea and the toac oil syndrome . Ann Intern Sled IA6 .I W :%30. anilide groups and then increased at day 28 . comet-Reino 11 Immune systems disorders associmed wish adult-,d Similarly, heated linoleic acid anilide groups showed a cocking e il . i n Berlin A, ewe J . Draper NW, Smith E61B, Spcunco F. decrease in the T cells at day 7. Among T cell subsets. T ads . LNEP. ILO . WHO Intemaliunal Pragamme cn Chemical Safety helper cells did not show any change, whereas T suppressor ([PCs)'. Commission of the European Communities Immunm¢sicology . Derdreche staniva, S'ttcn, 1987-316-89 . numbers decreased at days 1 and 7, but returned to control 6. Posadu do la Pact . 13.,6 IA . K11Mmmc ESL d a1 . talc cueof Icsk levels at day 28 . Our data appear to be consistent with the oindrl sm :1 dSen that the aeliolegical agent persisted in mI saliva alterations in T cell numbers recorded in patients with the for ap 1e one near . F-J Chem Tceoal 1989 .8117-2i . a. Pasada de In Pox S7 . Colm M, Kilboume E M . e t a1. Toxic-,,l syndrome: toxic oil syndrome (40). Our data further suggest that linoleic t oleic reports otrocated urh the ITH ail refinery in Seeilla . Foal Chem acid anilide could be more toxic than acid anilide and Tr.viccl Is%7ta:g;_9o. the changes observed appear to be more pronounced in the %. Kammuller ME, Bloksma N, Seines W . Chemical-induced einoimmure heated anilide groups, and Spanish tonic oit syndm,rr: tacos an ,d, iloins and related c_p-d,. Cain Toxieul 1988'26:157-74, 9. Altenk',eh H. Slalteoburg-Didinger G. Voeppel C . The neurotoxicolapi- cal a,p¢IS cr 1he bone ad syndrome ITOSI in Spain . Toxicol 19%8:492 Conclusions III. These studies mist several considerations about (lie fen RAL Kephert GM . Posada de la Pox hL et al. Panicipalion of mophil in the tonic oit syndrome . Cain E.ap 1--A 1990.82.313-1, of etiology the toxic oil syndrome. First, the background 11 . K :lbourne Ehl. Bemen IT Jr, Precda de la Pa, dL ci al . Chemical studies of vascular toxins (allylamine and beta-aminopropi- rr,hle, of p,ahngenicity of oils slated to the Ionic ail sOiireme onitrile) clearly indicate that vascular cells tin this case, the epidemic in Spain. Am J Epidemiol 1988 :127 :1_210-'_7. C . Starqucl A. Larraco V, Die, 1L. et al . lmmunogenicav of fury acid medial smooth muscle cell) may be subject to synergistic anilidoa in mbbirs and the palhogenu¢ etthe Spanish mxic oil syndrome . actions of toxins. This raises the possibility that several EaperienlSt19841d1977-80 . toxins found in the toxic oil syndrome could be necessary to 13 . Ccii,C .Oonov anrrenoP.}tuciciaAid. Lipogenesisinliver,tacoand J cause injury to blood vessels . In the case of the toxic oil adipose Mean, cross fed with ofeeylanifide . Biochem 1913:212:139-44. 14 . 3lasaa R . Sane P, Flare, I . Pastor T . Repeno S1 . Enrol of troy raid syndrome, however, it appears that endothelial cells rather ro iste r an the immune system of the Swiss mcuu : abnormalities in me than smooth muscle cells are the target . vnthesis of Immenodohulins iahvq . Pwscnled a1 the Mecling of the V Our laboratory has focused its preliminary investigations Inrmnunnal Congress of Toxicology. Brighton. England : Taylor & Francis . 1359 :132 . syndrome on the study the fatty of experimental toxic oil of 15 . Rodnca J . vAles SL Mayo L Pesmna A.II al. Neurmosiciry effuuy add acid anilides for the reasons detailed in this report . Man, anilides In rabbit,, Lancet 1983 :1 :414-6 . other toxic chemicals including esters of chloropropanediol 16 . Sore, A. VVeda 911. Gucia-Harreno P-and Munieio 9N . Toxic oil yWmme . Spain : erect orotcoylenilide nn the rotoaw of Wlysaturaled (40 have been implicated in the syndrome (8 .42-45). In f.o, aoin. :rod Lpid peronida0nn in nls- arch Envirvn Conlam Toxicol addition, the toxicity of rapeseed oil itself or its component. 19%5 :14 :131-6 . erucic acid, has been extensively studied for many years 17 . Caviar K. Hr,rsith RM . and Bcor Pl . Nlylamine :md ffaminopmpioni . v; (46-50) and was initially suggested as a toxic etiologic agent rilcinduced radar ininry : ate in visa and in sire a idy. Toxicol Appl Phi--l1991hI01:288-302 . in the toxic oil syndrome (51) . We suggest the possibility . I8 . Bon, PJ. Slaskn 51T. Reynelds ES. Allylamine cardiemxicily: I . Se therefore, that any of these toxic candidates could be acting quencc of pulh,,q,c events . Toxicol April Phannacol 19'.9:50:581-92. synergistically with others in the toxic oil syndrome . 1'7 . Latish IL Al en JR . Prix WCW. Myocardol fibrosis and smaNh mmdc ¢I1 hypcrpluia in .runny uneries of allylamine-fed rain . Am J Pathol Our data on the fatty acid anilides strongly suggest that . 1972 :66 :225-40 . these toxic compounds induce immunologic alterations that 213 . I.:Jkh 11 . C,-,ay artery hyolinosis in r ns fed allylamine. Exp Slat may be similar to those that underlie the toxic Oil syndrome. Paitn1 1969 :10 :14-'_6 .
1828 RODRI=.TAL. 14CC Vol. Ie . No. 7 SYNERI lSITC VASCULKR TOXINS IN TOXIC OIL SYNDROME Decem000 fOOc182s_I
'.I . Id amps K . Groesman SL. Cu.t LR, Allyl