Mutation Research, 239 (1990) 181-187

181

Elsevier MUTREV 07290

iNTERNATiONAL COMMISSION FOR PROTECTION AGAINST ENVIRONMENTAL MUTAGENS AND CARClNOGENS

ICPEMC Working Paper 7/1/3

Animal studies suggesting involvement of mutagen/carcinogen exposure in atherosclerosis Keiji W a k a b a y a s h i Carcinogenesis Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 (Japan)

(Accepted 17 May 1990)

Keywords: Mutagen/carcinogen;Radiation;Atherosclerosis;Animalstudies

Summary It is very important to elucidate the causative agents of atherosclerosis because coronary heart disease and cerebrovascular disease are the main causes of death in the developed countries. The evidence for a monoclonal origin of atherosclerotic plaques in humans prompted the study of the involvement of mutagens/carcinogens in the development of atherosclerosis. Polycyclic hydrocarbons, including 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene, were shown to act as initiators a n d / o r accelerators in atherosclerotic plaque formation in the chicken, pigeon and mouse. Radiation and oxygen radicals were also demonstrated to be involved in the development of atherosclerosis in animals.

Cancer, coronary heart disease and cerebrovascular disease are the main causes of death in the U.S.A., Western Europe and Japan. The development of coronary heart disease and cerebrovascular disease is mostly due to atherosclerosis of the arteries. The atherosclerotic plaque consists of proliferated smooth muscle cells infiltrated with

Correspondence: Dr. J.D. Jansen, MedicalBiologicalLaboratory TNO, P.O, Box 45, 2280 Rijswijk(The Netherlands). ICPEMC is affiliatedwith the International Association of EnvironmentalMutagenSocieties(IAEMS)and the Institutde la Vie.

macrophages and lipid deposits, collagen, elastin and glycosaminoglycans. By examining the isoenzymes of glucose-6-phosphate dehydrogenase, it has been shown that atherosclerotic plaques in humans are monoclonal in origin (Benditt and Benditt, 1973). In animal experiments, chemical carcinogens, radiation and oncogenic viruses were also shown to be involved in the development of atherosclerotic plaque (Lindsay et al., 1962a,b; Penn, 1989). Furthermore, DNA samples from human coronary artery plaques were recently demonstrated to transform NIH3T3 cells in the focus-forming assay (Penn et al., 1986). These results suggest that atherosclerotic plaques are

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presumably benign smooth muscle cell tumors formed in the arterial wall. However, the role of environmental factors, such as mutagens/carcinogens, radiation and oncogenic viruses, i n atherosclerosis has not yet been fully elucidated, although cigarette smoking, hypercholesterolemia and hypertension are known risk factors for atherosclerosis (Solberg et al., 1980). In this paper, I will review animal studies suggesting that environmental mutagens/carcinogens may be involved in atherosclerosis. In addition, the effect of radiation on the formation of atherosclerotic plaques in animals is also briefly described. Animal experiments showing the effect of mutagens/carcinogens on the formation of atherosclerotic plaques Chickens Atherosclerotic plaques have been found to develop spontaneously in the abdominal aorta of the chicken (Paterson, 1967). Using this animal, the effects of chemical carcinogens on plaque formation were first tested by Albert et al. (1977). Two carcinogenic polycyclic hydrocarbons, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), were dissolved in dimethyl sulfoxide and injected into the pectoral muscle of SC-strain chickens at concentrations of 25 m g / kg/injection and 50 mg/kg/injection, respectively. Animals received the carcinogens weekly from 4 to 22 weeks of age. After treatment with the carcinogens, the aorta was excised and the anterior wall was opened lengthwise. Then the aorta was sectioned transversely into 3-mm segments. After paraffin embedding, histological sections stained by the Verhoff-Van Gieson procedure or with hematoxylin and eosin were prepared. The cross-sectional area of the atherosclerotic plaque on each segment was measured microscopicaUy. Atherosclerotic plaques were observed only in the abdominal aorta, almost exclusively above the iliac trifurcation, in both experimental and control animals. The plaque lesions, located between the internal elastic lamina and the endothelium, were fibrous with a substantial cellular component and little evidence of extraceUular lipid accumulation

or foam cells. The areas of plaques in the crosssections of aortas from chickens treated with DMBA and B[a]P were significantly larger than those from non-treated animals (Albert et al., 1977). DMBA was more potent than B[a]P in enlarging plaque size. Furthermore, incorporation of [3H]thymidine into the aorta was clearly greater in the plaques than in the medial cells. The levels of serum cholesterol in the animals were not affected by treatment with the chemical carcinogens. Administration of 0.1, 1 and 10 mg of D M B A / kg/injection weekly for 20 weeks into SC-strain chickens showed a dose-dependent increase in atherosclerotic lesion size (Bond et al., 1981). A similar result was obtained following treatment with B[a]P. On the other hand, weekly administration of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), at a dose of 0.1 mg/kg/injection following treatment with a single dose of DMBA or B[a]P did not enhance the formation of atherosclerotic lesions (Bond et al., 1981). The effects of various doses (5, 10 and 20 mg/kg/injection) of DMBA on the development of atherosclerotic lesions in male white leghorn chickens were also investigated by Penn et al. (1981a). After i.m. administration from 5 to 20 weeks of age, plaque cross-sectional areas increased in a dose-dependent manner 7-11-fold over control animals. However, plaque frequency in animals treated with DMBA was similar to that of non-treated animals (Penn et al., 1981a). Another study investigating age-dependent changes in frequency, size and proliferation of arterial lesions in cockerels again showed that DMBA had no effect on the frequency of atherosclerotic plaque formation (Penn et al., 1981b). The sizes of plaques in animals treated with DMBA from 4 to 8 weeks of age were almost the same as those in control animals at 20 weeks of age. No difference was observed histologically or ultrastrncturally in plaques from treated and non-treated cockerels (Batastini and Penn, 1984). From the above observations, it is suggested that treatment with polycyclic hydrocarbons, including DMBA and B[a]P, accelerates the growth rate of preexisting spontaneous plaques in the abdominal aorta but does not induce the formation of new plaques. That is, DMBA and B[a]P presumably act as accelerators rather than as initiators of

183 atherosclerotic plaque development in the abdominal aorta of cockerels. This suggestion was further confirmed in an experiment testing the relationship between mutagenic and carcinogenic potency and plaque-forming ability in the abdominal aorta using a variety of mutagens/carcinogens (Penn and Snyder, 1988). Plaque sizes in cockerels treated with B[a]P, benzo[e]pyrene (B[e]P), dibenz[a,c]anthracene, 3-methylcholanthrene and dibenz[ a, h ]anthracene were clearly larger (7.8- to 13.5-fold) than those in cockerels treated with only dimethyl sulfoxide as a solvent. The potency of dibenz[ a, h]anthracene was the same as that of DMBA. 2-Acetylaminofluorene (2-AAF) slightly accelerated plaque formation, but N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and anthracene both showed httle effect. The order of the plaque-stimulating potencies of these mutagens/carcinogens was: DMBA = dibenz[a, h]anthracene > 3-methylcholanthrene > dibenz[a,c]anthracene > B[e]P > B[a]P >> 2-AAF > M N N G > anthracene. Thus, no relation between mutagenic and carcinogenic potency and atherosclerotic plaque-forming ability was evident. These mutagens/carcinogens do act as accelerators in plaque formation. On the other hand, sequential treatment with an initiator and a promoter was found to induce focal proliferation of intimal smooth muscle cells in the thoracic aorta of chickens. Once weekly from 4 to 6 weeks of age, SC-strain chickens received an intraperitoneal injection of DMBA (10 mg/kg/injection), followed by a twice weekly injection of methoxamine (5 mg/kg/injection) from 7 to 27 weeks of age. Methoxamine is an al-selective adrenergic agonist and is reported to induce ornithine decarboxylase activity in the aorta (Majesky et al., 1982). The sequential treatment with DMBA and methoxamine produced a greater number of small mound-like lesions, detected by scanning electron microscopy, in the thoracic aorta than treatment with DMBA or methoxamine alone (Majesky et al., 1985). All the lesions showed similar sizes (50-100 /~m wide and 100-150 gm long), and were composed of densely packed modified smooth muscle cells without any evidence of lipid accumulation. These lesions were not observed in segments of thoracic aorta from chickens treated with only DMBA (10 mg/kg/injection)

for 23 weeks nor in untreated or vehicle-treated chickens. Contrary to this, the combined administration of DMBA and methoxamine did not greatly enhance plaque formation in the abdominal aorta. Of course, injections of DMBA for 23 weeks markedly stimulated the growth of plaques in the abdominal aorta as reported in other papers. In addition to the effect of mutagens/ carcinogens, especially polycyclic hydrocarbons, on the development of atherosclerotic plaques in the chicken aorta as stated above, cytochrome P-450-dependent monooxygenation and bioactivation of the polycyclic hydrocarbons DMBA and B[a]P in the aorta were also studied (Bond et al., 1980). Chicken aortic homogenates were found to contain enzymes which form the oxidized products of DMBA and B[a]P and generate the reactive intermediates that bind covalently to calf thymus DNA. An $9 fraction prepared from the aortic homogenates catalyzed the conversion of these polycyclic hydrocarbons to mutagens detected using Salmonella typhimurium TA98 and TA1538. Furthermore, chicken aortic tissues contain sulfotransferase and UDP-glucuronosyltransferase, which catalyze the sulfation and glucuronidation of 3-hydroxy-B[a]P, respectively (Yang et al., 1986a,b). Human thoracic aorta tissue also has monooxygenase activity (Juchau et al., 1976) as do cultured human fetal aortic smooth muscle cells (Bond et al., 1979).

Pigeons Atherosclerosis-susceptible White Carneau pigeons develop spontaneous aortic lesions (Clarkson et al., 1959). Cytochrome P-450-dependent monooxygenase levels in aortic and hepatic tissues of these pigeons were greatly increased by treatment with cytochrome P-450 inducers (Majesky, 1983). In atherosclerosis-resistant Show Racer pigeons, the inducibility was much lower. Atherosclerotic lesions were induced or enhanced in White Carneau pigeons treated with B[a]P, 3methylcholanthrene and DMBA, but not with B[e]P and 2,4,6-trichlorophenol (Revis et al., 1984). Chemicals were dissolved in corn oil and injected weekly into the pectoral muscles of male pigeons at doses of 0.1, 10 or 100 mg/kg body weight for 3 or 6 months. The number and sizes of aortic atherosclerotic plaques were significantly

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larger in pigeons treated with B[a]P and 3-methylcholanthrene. DMBA clearly enhanced the development of coronary arterial plaques. On the other hand, DMBA was unable to induce or enhance the growth of aortic atherosclerotic plaques, in contrast to its performance in the chicken. The coronary arterial plaques were increased in pigeons treated with 3-methylcholanthrene at almost the same incidence as after treatment with DMBA, and also increased with B[a]P, though at a lower incidence than with DMBA. In addition, these 3 polycyclic hydrocarbons induced plaques in the carotid and pectoral arteries. N o relationship was observed between the increase in atherosclerosis and either arterial pressure, plasma cholesterol or the L D L / H D L cholesterol ratio in this experiment (Revis et al., 1984). Mice The effect of 3-methylcholanthrene on the development of atherosclerosis was studied in 2 congenic mouse strains, AKXL-38a and AKXL-38, which differ at the locus Ah (Paigen et al., 1985, 1986). The Ah-responsive strain, AKXL-38a, is more susceptible to cancer induced by 3-methylcholanthrene than the Ah-non-responsive strain, AKXL-38. The 2 mouse strains were fed a mixed diet, consisting of an atherogenic diet and breeder chow at a ratio of 1 : 3 for 14 weeks. The atherogenic diet contains 15% fat, 1.25% cholesterol and 0.5% sodium cholate. 3-Methylcholanthrene was injected intraperitoneally at a dose of l / ~ g / m o u s e on days 0, 2 and 7. The heart and the upper section of the aorta were removed, and aortic lesions were evaluated using oil red O and hematoxylin light green staining. The number of lesions and the lesion area were increased in both strains of mice treated with 3-methylcholanthrene, but the effect was larger in the Ah-responsive strain (Paigen et al., 1986). Treatment with 3-methylcholanthrene did not affect the levels of high-density lipoprotein. In addition to the above study, early experiments by White et al. (1941, 1942) indicated that treatment with 3-methylcholanthrene induced lesions in the aorta and other large arteries in Dilute Brown (dba) strain mice fed a low-cystine diet. The chemical was painted on the skin of the mice on alternate days between 20 and 70 times.

Leukemia was induced at a high incidence in treated mice ingesting control chow or a highcystine diet. On the other hand, the incidence of leukemia was markedly reduced in the mice which were fed the low-cystine diet, but atherosclerotic lesions were frequently observed in these animals. When the amount of cystine in the diet was limited, weight gain of the mice was slowed. Recently, p-hydrazinobenzoic acid, which is present in the cultivated mushroom, Agaricus bisporus (Chauhan et al., 1984), was demonstrated to induce smooth muscle cell tumors of the aorta and large arteries (McManus et al., 1987). p-Hydrazinobenzoic acid-hydrochloride salt was dissolved in drinking water at a concentration of 0.125% and given to Swiss mice for life. Tumors developed in 14% of females and 42% of males in the mice treated with the chemical, and in none of the female and 4% of the male controls. The aortic tumors were characterized as leiomyomas and leiomyosarcomas. These results suggest that this hydrazine derivative would form atherosclerotic plaques in animals under some conditions. Various kinds of carcinogenic heterocyclic amines are present in cooked food. Four heterocyclic amines, 2-amino-6-methyldipyrido[1,2a : 3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a : 3',2'-d]imidazole (Glu-P-2), 2-amino-9Hpyrido[2,3-b]indole (AaC) and 2-amino-3-methyl9H-pyrido[2,3-b]indole (MeAaC), were reported to produce hemangioendothelial sarcomas in CDF 1 mice when these compounds were administered in the diet (Sugimura, 1986). Furthermore, C D F 1 mice fed a diet containing 0.05% Glu-P-1 for 4 months developed calcification and angioproliferation in the epicardium of the right ventricle of the heart (Takayama et al., 1987). Thus, it is suggested that some of the heterocyclic amines m a y be involved in the development of atheroclerosis in animals. Rats F344 male rats received an intrarenal injection of carcinogenic nickel subsulfide (Ni3S2) at a dose of 5 rag/rat, then were killed after 7 or 9 weeks. Atherosclerotic lesions were clearly observed in the aorta and large arteries of rats. Erythrocytosis, glomerulomegaly, hyperplasia of mesangial cells and increased deposition of mesangial matrix were

185 also induced in nickel subsulfide-treated rats. Hypertension and hyperlipidemia were not attributable to atherosclerotic lesions induced by nickel subsulfide (Hopfer et al., 1984). The mechanism by which nickel subsulfide induces atherosclerotic lesions has not yet been elucidated. Rabbits It has been suggested that low-density lipoprotein modified by oxidation could contribute to foam cell formation during atherosclerosis (Steinbrecher et al., 1984). Kita et al. (1987) demonstrated that probucol, an antioxidant, inhibited the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic ( W H H L ) rabbits which are an animal model for human familial hypercholesterolemia. Furthermore, this inhibition was shown to be due to limiting oxidative modification of low-density lipoprotein and foam cell transformation of macrophages. Thus, oxygen radicals may play an important role in the development of atherosclerosis. The effect of radiation on the formation of atherosclerotic plaques in animals

Two breeds of pigeons, atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer, were used to test the effect of radiation on atherosclerosis development (Artom et al., 1965). Prevalence and severity of atherosclerosis in the coronary arteries of White Carneau pigeons fed a 0.1% cholesterol diet increased by wholebody treatment with large cumulative doses of X-irradiation (5500 R). N o effect of X-irradiation was observed on atherosclerotic lesions in the thoracic aorta of White Carneau pigeons fed cholesterol-supplemented diets. On the other hand, X-irradiation increased the development of atherosclerosis in the thoracic aorta of Show Racer pigeons fed a 1% cholesterol diet, but not in the coronary arteries. Male albino Wistar rats, which were fed a high-fat diet including 2% cholesterol, received X-irradiation at a total of 2500 R to the thorax. The rats were autopsied 8-15 weeks after irradiation. The number and degree of atherosclerotic lesions in the coronary and main pulmonary arteries and also in the endocardium were signifi-

cantly larger in irradiated rats than in non-irradiated rats (Gold, 1961). Segments of the abdominal aortas of young adult mongrel dogs were irradiated with X-rays at doses of 1500-5500 R. Much more severe aortic lesions were observed in the irradiated sites than in non-irradiated sites. Histologically, the atherosclerotic lesions that developed after irradiation were very similar to those occurring spontaneously in old dogs (Lindsay et al., 1962a). Similarly, aortic atherosclerosis was induced by electron irradiation in dogs (Lindsay et al., 1962b). X-irradiation was also demonstrated to enhance the development of atherosclerosis in locally irradiated carotid arteries of hypercholesterolemic rabbits (Lamberts and de Boer, 1963; Konings et al., 1980). When rabbits fed a 0.5% cholesterol diet were treated with X-irradiation, atherosclerotic lesions appeared within 6 weeks. In the case of non-irradiated hypercholesterolemic rabbits ingesting 0.5 and 2% cholesterol, atheromas developed after 5 and 3 months, respectively (Konings et al., 1980). Thus, radiation may produce injury to the vascular wall which accelerates atherosclerosis. Furthermore, clinical radiotherapy probably enhances aortic lesions in humans. Discussion

The effect of mutagens/carcinogens on the formation of atherosclerotic plaques has been examined in chickens, pigeons, mice and rats. Among these animal models, the chicken has been used most extensively, because it is very susceptible to atherosclerosis and the characteristics of the lesions are very similar to those in human arteries. Furthermore, the association of viruses with aortic lesions was also investigated in chickens (Fabricant et al., 1978). Mice and rats rarely develop spontaneous atherosclerosis under normal conditions. Therefore, cholesterol was added to the diet in order to investigate atherosclerotic lesions in mouse experiments. Polycyclic hydrocarbons such as DMBA, B[ a ]P and 3-methylcholanthrene were clearly shown to act as initiators a n d / o r accelerators in the development of atherosclerotic plaques in many animal experiments. However, it is not clear whether somatic mutations in the smooth muscle cells of

186 the aorta occurred or not, and which effects of t h e s e c h e m i c a l s a r e r e l a t e d to t h e f o r m a t i o n o f atherosclerotic plaques. In addition, knowledge of t h e effect o f o t h e r m u t a g e n s / c a r c i n o g e n s on a t h e r o s c l e r o s i s d e v e l o p m e n t is v e r y l i m i t e d , although a variety of mutagens/carcinogens are d i s t r i b u t e d in o u r e n v i r o n m e n t . T h e a m o u n t s o f mutagens/carcinogens used experimentally are also l a r g e r t h a n t h o s e to w h i c h h u m a n s a r e exp o s e d in o r d i n a r y life. T h u s , t h e p r e s e n t s t a t u s o f r e s e a r c h o n t h e e f f e c t o f m u t a g e n s / c a r c i n o g e n s in t h e f o r m a t i o n o f a t h e r o s c l e r o t i c p l a q u e s is still preliminary, and the exact role of environmental mutagens/carcinogens in t h e d e v e l o p m e n t o f a t h e r o s c l e r o s i s in h u m a n s c a n n o t yet b e d e d u c e d . S i m i l a r to t h e p r o c e s s o f h u m a n c a n c e r d e v e l o p ment, various kinds of factors, including mutag e n s / c a r c i n o g e n s a n d r a d i a t i o n , are p r o b a b l y inv o l v e d in a t h e r o s c l e r o s i s in h u m a n s . I t r e m a i n s v e r y i m p o r t a n t to s t u d y t h e c o n t r i b u t i o n o f e a c h f a c t o r in h u m a n a t h e r o s c l e r o s i s , b e c a u s e c o r o n a r y h e a r t d i s e a s e a n d c e r e b r o v a s c u l a r d i s e a s e a r e as b i g a t h r e a t to h u m a n h e a l t h as c a n c e r .

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