Dietary and metabolic manipulations of the carcinogenic process: role of nucleotide pool imbalances in carcinogenesis

Perturbations in DNA and/or membranes are considered to be important for the carcinogenic process. A search for nutritional and metabolic means of disturbing the homeostasis of DNA and membranes revealed that nucleotide pools offer an exciting possibility. An imbalance in nucleotide pools can exert a two-pronged attack on both DNA and membranes. When given to rats, orotic acid, a precursor of pyrimidine nucleotides, results in an imbalance in nucleotide pools (an increase in uridine nucleotides and a decrease in inosine/adenine nucleotides), alterations in both DNA and membranes, and promotion of carcinogenesis in the liver initiated by chemical carcinogens. Agents such as adenine and allopurinol, which inhibit the metabolism of orotic acid and thereby decrease the formation of uridine nucleotides, and galactosamine, which traps uridine nucleotides, inhibited the promotional effects of orotic acid in the liver. These results suggested that orotic acid needs to be metabolized to uridine nucleotides and the creation of a subsequent imbalance in nucleotide pools is important for the promotional effects of orotic acid. To determine whether the creation of a nucleotide pool imbalance is a more general mechanism of tumor promotion, two lines of approach were investigated. One was to determine the effect of orotic acid on promotion of carcinogenesis in other organs, and the second approach was to determine how to induce nucleotide pool imbalances by means other than orotic acid administration. It is interesting to note that orotic acid promotes carcinogenesis in duodenum initiated by azoxymethane. Regarding the second approach, it became apparent that several metabolic disturbances result in increased orotic acid synthesis and alterations in nucleotide pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of cell death (apoptosis) in preneoplastic and neoplastic liver cells. A sequential study.

A sequential study was performed to investigate the occurrence of cell death in preneoplastic and neoplastic liver cells of F-344 rats. The animals were administered a single initiator dose of 1,2-dimethylhydrazine and were then subjected to a liver carcinogenesis promotion regimen, consisting of a diet containing 1% orotic acid. Cell death, morphologically similar to that described as apoptosis, was evident in foci of preneoplastic hepatocytes at 10 weeks after orotic acid feeding. An increased frequency of apoptotic bodies was observed in nodules, but not in the surrounding liver, 20 weeks after starting the dietary regimen, and in hepatocellular carcinomas that developed after 1 year of continuous promotion. Occurrence of this type of cell death was also observed in liver foci of rats subjected to two other promoting regimens, suggesting, thus, a possible relevance of apoptosis to the carcinogenic process in the liver.     

Is there more than one critical lesion relevant for experimental liver carcinogenesis?

The present study was designed to determine whether there is one or more than one critical lesion, induced by a carcinogen, relevant for initiation. The experimental approach consisted of administering a non-necrogenic dose of the carcinogen 1,2-dimethylhydrazine 2HCl (100 mg/kg, i.p.) to male Fischer 344 rats (120-140 g) and completing the initiation process by two different methods: (i) induction of liver cell proliferation by partial hepatectomy, or (ii) creation of hypomethylation in DNA by giving 5-azacytidine, an agent that inhibits DNA methylation. The initiated hepatocytes were assayed as gamma-glutamyltransferase positive foci. The rationale for the approach was based on the premise that the two methods used for completing the initiation step might give either the same or a different pattern in the incidence of initiated hepatocytes depending on whether one or more than one lesion was important for initiation, particularly if some of the lesions were allowed to repair before applying the cell proliferative stimulus or administering the 5-azacytidine. The results obtained indicated that 5-azacytidine facilitated the induction of the same number of foci whether given 12 or 96 hours after the carcinogen indicating that the critical lesion involved in this mode of initiation persisted up to at least 96 hours. In contrast, our earlier results showed that there was a reduction in the number of gamma-glutamyltransferase positive foci when partial hepatectomy was delayed beyond 24 hours after the carcinogen administration, indicating that the critical lesion involved in this mode of initiation has a half-life of not more than 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complementarity between two rat liver tumor promoters

A delay in the exposure of initiated rats to orotic acid (OA) beyond a specific time frame results in a progressive loss of promotional effect in liver carcinogenesis. The current study was designed to ascertain whether the loss of promotional effect could be counteracted by pre-exposing the initiated animals to other rat liver promoting regimens such as a diet deficient in choline (CD). Male Fischer 344 rats (150 g) were initiated with diethylnitrosamine (200 mg/kg, ip); 1 week later they were given either a CD diet or a CD diet supplemented with choline for 5 weeks. Animals from these two groups were then fed either a 1% OA diet or the basal diet for another 20 weeks. The results indicated that the loss of OAs promotion efficacy from delaying the start of the promoting regimen can be counteracted by pre-exposing the initiated rats to a CD diet. Thus in rats exposed to OA from the first week of initiation, 7% of the liver developed as nodular areas, whereas only 0.8% of liver was nodular when OA feeding was delayed by 5 weeks. This loss was abolished when initiated rats were fed a CD diet for 5 weeks prior to feeding OA for 20 weeks. These results suggest that in a rat liver tumor promotion model, two tumor promoters, OA and CD, show some degree of complementarity when given sequentially.     

Influence of level of dietary protein on tryptophan-induced promotional activity in induction of gamma-glutamyl transpeptidase-positive foci of rat liver

The influence of varying the dietary protein content on the emergence of gamma-glutamyl transpeptidase (GGT)-positive foci in the livers of male rats fed elevated (2%) L-tryptophan (TRP) after being exposed to a hepatocarcinogen was investigated. Subtotal hepatectomies were performed, and 18 hr later the rats were treated with diethylnitrosamine (30 mg/kg). Ten days later four dietary groups were followed for 10 weeks: (1) control diet containing 21% protein (C); (2) control diet containing 5.3% protein (C-LP); (3) C + TRP; and (4) C-LP + TRP. Rats fed the C-LP diet developed heavier livers but fewer and smaller GGT + foci than did rats fed the C diet. Rats fed elevated TRP diets (C + TRP and C-LP + TRP) developed more and larger GGT + foci than did rats fed the regular control diets (C and C-LP), indicating that the promotional effect of elevated dietary TRP was similar at the two levels of dietary protein.     

Cingulate lesions and behavioral adaptation to amino acid imbalanced diets

The effect of anterior cingulate cortex lesions on dietary intake and adaptation of disproportionate amounts of amino acids was examined. Rats with bilateral electrolytic lesions in the anterior cingulate cortex and sham-operated rats were fed, in turn, amino acid basal, imbalanced or devoid diets involving threonine and isoleucine as the growth limiting amino acids, and then a low protein (6% casein) followed by a high protein (75% casein) diet. Lesions of the anterior cingulate cortex did not prevent the initial depression in food intake of the amino acid imbalanced diets, but shortened the duration of anorexia associated with dietary amino acid imbalances. Cingulate lesions did not influence the food intake of rats fed amino acid devoid diets. When switched from a low protein to a high protein diet, animals bearing lesions and sham-operated controls reduced markedly their initial food intake and adapted to the high protein diet in similar manner. It was concluded that the initial food intake depression associated with a dietary amino acid imbalance is a direct response to postingestive cues which influence food intake. Moreover, that the difference in adaptive intakes of the cingulate cortex lesioned animals who ingested a diet of imbalanced amino acids or of high protein, indicates that separate mechanisms act to control food intake of animals fed diets containing imbalanced amino acid mixtures or diets with excessive amounts of protein.     

Diuron lacks promoting potential in a rat liver bioassay

The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.     

Orotic acid overproduction in experimental cirrhosis of rats

Ammonia clearance, portal blood ammonia, and amino acid concentrations were studied during induction of cirrhosis by carbon tetrachloride in rats. Exposure to CCl4 vapors twice weekly for 7-16 weeks doubled orotic acid excretion. If exposure was discontinued for 7 days, the orotic acid excretion decreased despite the presence of cirrhosis proven histologically. Replacement of dietary casein with soybean protein eliminated the CCl4-induced orotic aciduria in growing rats but not in adults. Supplementation of casein with 1.5% arginine did not prevent CCl4-induced orotic aciduria. [14C]Orotate uptake into RNA and DNA of liver was not impaired. Perfusion of livers of cirrhotic animals with ammonia concentrations between 0.2 and 3.0 mM revealed no significant decreases in urea synthesis rates due to cirrhosis and no increase in the tendency to make orotic acid at a given ammonia concentration. However, ammonia uptake by cirrhotic livers was significantly reduced, resulting in higher ammonia concentrations in the effluent when there was moderate-to-severe cirrhosis. Portal blood samples taken from rats exposed to CCl4 had higher ammonia concentrations as cirrhosis worsened. The results lend support to the "intact hepatocyte" hypothesis of cirrhosis which attributes metabolic abnormalities to intrahepatic shunts.     

Overeating, overweight and obesity induced by an unpreferred diet

Rats fed diets containing 50-71% added water (liquid diets) eat more energy and gain more weight than rats fed the same diets without added water (solid diets). The present experiments examined the effects of making a liquid diet less palatable. The first experiment examined the effects of sucrose octaacetate on diet preference. Rats, given a choice of a liquid diet containing 0.5% sucrose octaacetate and a plain solid diet, preferred the plain solid diet for three weeks. When the concentration of sucrose octaacetate was reduced to 0.05%, the rats did not show a reliable preference for either the sucrose octaacetate liquid or plain dry diet. In subsequent experiments, each rat was given only one diet at a time. In the second experiment, rats were fed 0.5% sucrose octaacetate liquid diet for three weeks followed by 0.05% sucrose octaacetate liquid diet for another four weeks. The rats fed the sucrose octaacetate liquid diet overate and became obese compared to the rats fed plain solid diet throughout. In the third experiment, rats fed 0.5% sucrose octaacetate liquid diet for six weeks became obese compared to rats fed plain solid diet throughout. Thus, the overeating and obesity induced by liquid diets cannot be attributed solely to their high palatability.     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

The Natural History of Neoplasia: Newer Insights Into an Old Problem

The stages of initiation and promotion in the natural history of epidermal carcinogenesis have been known for many years. Recently, experimental systems other than skin have been shown to exhibit similar, if not completely analogous, stages in the natural history of neoplasia. In particular, the demonstration by Peraino and his associates that phenobarbital may enhance the production of hepatomas by a relatively subcarcinogenic dose of acetylaminofluorene was one of the first demonstrations of stages occurring in an extraepidermal neoplasm. Studies reported in this paper have demonstrated that administration of phenobarbital (0.05% in the diet) for 6 months following a single dose of diethylnitrosamine (5 to 10 mg/kg) given within 24 hours after partial hepatectomy resulted in a marked increase in the number of enzyme-altered foci in the liver as well as in the production of hepatocellular carcinomas. This was compared to animals receiving only a single dose of diethylnitrosamine following partial hepatectomy with no further treatment, in which only a relatively small number of foci were evident in the absence of phenobarbital feeding. Using three different enzyme markers, a distinct degree of phenotypic heterogeneity of the enzyme-altered foci in liver was demonstrated. These studies have shown that liver carcinogensis can be readily divided into two stages: a) initiation by a single dose of diethylnitrosamine following partial hepatectomy and b) promotion by the continuous feeding of phenobarbital. Furthermore, the immediate progeny of the initiated cells, the enzyme-altered focus, may be recognized by suitable microscopic means prior to the formation of gross lesions as required in the skin system. These initiated cell populations exhibit a degree of biochemical heterogeneity which reflects that seen in fully developed hepatic neoplasms, suggesting that promotion and progression in this system does not significantly alter the basic biochemical characteristics of the initiated cell.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes

Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.     

To the mechanism of enhancing the activity of serum and hepatic sorbitol dehydrogenase in rats fed excess sweetener sorbitol

One-month rats were randomized to a diet containing sorbitol (SOR) (54%), saccharose (SACCH) (54%) or that containing SOR (27%) and SACCH (27%). They were kept on the diets for 1 or 3 months. At the age of 2 and 4 months, the rats were returned to the routine diet of a vivarium. The experiments were terminated when the rats were aged 2, 4, and 7 months. In all seasons, the monthly diets containing 27% and 54% of SOR increased the serum activity of sortibol dehydrogenase (SDH) by several and tens of times. The 3-month diet produced the same effect. It suddenly turned out that the 54% SACCH diet also increased SDH activity, but to a lesser extent than a combination of 27 SOR and 27% SACCH, and by tens of times less than the 54% SOR diet. Comparison of three diets showed the same relationship between the increases in SDH activity in the liver. With excess dietary SOR, a very high increase was first found in serum and hepatic SOD activity by an induction mechanism.     

Effects of the peroxisome proliferator di(2-ethylhexyl)phthalate on enzymes in rat liver and on carcinogen-induced liver altered foci in comparison to the promoter phenobarbital

The livers of rats given either the peroxisome proliferating hepatocarcinogen di(2-ethylhexyl)phthalate (DEHP) following initiation by 2-acetylaminofluorene (AAF) or the neoplasm promoter phenobarbital (PB) were studied for changes in 8 histochemical properties. Male F344 rats were fed 200 ppm AAF for 7 weeks to induce hepatocellular altered foci, and were then fed diets containing either no chemical, 12,000 ppm DEHP or 500 ppm PB for 24 weeks. In hepatocytes, DEHP increased alkaline phosphatase activity throughout the lobule, but reduced gamma-glutamyltransferase (GGT) activity in periportal hepatocytes. PB, in contrast, increased GGT activity in periportal hepatocytes. In foci that were induced by AAF, DEHP reduced the histochemical activity of GGT and did not increase the number, mean volume or volume % of foci detected by deficiencies in iron storage, glucose-6-phosphatase, adenosine triphosphatase or fibronectin. PB enhanced the expression of all 8 phenotypic abnormalities in foci such that either more profiles were detected or the area of foci was increased.     

Age-related changes in respiration coupled to phosphorylation. I. Hepatic mitochondria

Age-related changes affecting mitochondrial adenine nucleotide metabolism may underlie age-related decreases in hepatic metabolic activities. Oxidative activity coupled with phosphorylation, the apparent Km and Vmax of the adenine nucleotide translocase (AdNT), the adenine nucleotide pool size and membrane lipid composition were determined for hepatic mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. The age-related decreases in state 3 respiration supported by NAD-linked substrates were 2-4-fold greater than that of an FAD-linked substrate. The 32% (P less than 0.05) decrease in the AdNT Vmax calculated for the aged rats was accompanied by a 17% decrease in the AdNT Km. The exchangeable pool of adenine nucleotides in mitochondria from aged rats was 72% (P less than 0.05) that in the young rats. While the age-related increase in the cholesterol/phospholipid-Pi ratio and changes in the phospholipid head group pattern were not significant, the overall change in the fatty acid pattern effected a 20% (P less than 0.05) decrease in the n-6/n-3 fatty acid ratio. These data suggest that the reduced Vmax of the AdNT is a consequence of a diminished pool of exchangeable adenine nucleotides. The lower AdNT velocity may reflect the effect of changes in the lipid environment of the membrane in which it is embedded. The major shifts in these parameters occurred during the second year of life.     

Changes in myocardial pyrimidine nucleotide levels following repeated injections of isoproterenol in rats

Changes which might lead to the initiation of cardiac hypertrophy include possible variations in the dynamics of nucleotides. In the experiments reported in this paper, changes in the pool sizes of adenine, uracil and cytosine nucleotides were observed during the initial phase of cardiac overload.Repeated subcutaneous injections of isoproterenol (ISO) (5 mg·kg^–1 body weight, s.c.) were performed so as to produce symmetric cardiomegaly in rats. Under these conditions, the dry weight of the heart, on the fifth day of dialy injections of ISO, had increased by 43% and the RNA concentration by 39%. There was no significant change in the DNA concentration. No further changes in weight or in DNA and RNA concentrations were recorded from the fifth to the tenth day of treatment.Changes in UTP and ATP were carefully monitored during the first days following ISO application. The levels of both nucleotides decreased sharply at first. The ATP level remained below the control value for at least 48 h while the UTP level was rapidly restored and a further increase occurred resulting in a maximal enlargement of 82% after the 12th h. At the same time, the uracil nucleotide pool and the cytosine nucleotide pool had increased by 76% and 101%, respectively, while the adenine nucleotide content of the myocardium remained 15% below control level. Repeated injections of ISO induced effects on ATP and UTP levels which were similar in direction but attenuated.The significance of an increase in the pyrimidine nucleotide pools in relation to nucleic acid synthesis is discussed.     

Increased incidence of hepatic foci and nodules in rats given one or two doses of 1,2-dibromoethane

Livers of male Sprague-Dawley rats were evaluated for foci and nodules 90 days and 16 months after one or two oral doses of 1,2-dibromoethane (DBE). Rats (190 g) were given the following oral treatments. Controls received corn oil (2.5 ml/kg) at 0 and 24 hr. DBE X 1 received corn oil at 0 hr and 75 mg DBE/kg at 24 hr. DBE X 2 received 75 mg DBE/kg at 0 and 24 hr. All rats underwent a two-thirds partial hepatectomy at 28-29 hr, a one-third partial hepatectomy at 90 days, and were given 0.05% phenobarbital in drinking water for 4 months beginning at 1 yr. DBE was given to the DBE X 2 group twice within 24 hr because the compound is a hepatocyte mitogen. At 90 days, no appreciable changes were evident in any group. At 16 months, the incidence of nodules in the DBE X 2 group (25 of 41) was double that of the DBE X 1 group (12 of 38) (p less than 0.01) and triple that of the control group (6 of 34) (p less than 0.0001). Both the DBE X 1 and the DBE X 2 groups had higher incidences of eosinophilic foci, and also higher numbers, larger sizes, and larger areas of gamma-glutamyltranspeptidase-positive foci than the control group. These results demonstrate for the first time that hepatocyte foci and nodules are initiated by limited exposure of animals to DBE.     

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.     

Myocardial ultrastructure of rats fed high and low erucic acid rapeseed oils

Ultrastructural study was conducted on ventricular muscle obtained from male rats of the Sprague-Dawley strain after 18 and 28 weeks of feeding diets containing 20% (w/w) soybean oil, low erucic acid rapeseed oil, or high erucic acid rapeseed oil. Myocardium of the rats fed soybean oil-containing diet was apparently normal despite high content of oil in the diet. Long-term feeding of high erucic acid rapeseed oil resulted in alteration of mitochondrial morphology, disorganization of myofibrils, and degeneration or necrosis of the cardiac muscle fibers. Macrophages were observed near the degenerating muscle cells but these cells did not exhibit accumulation of lipid droplets. Old lesions were characterized by an increase in collagen fibrils. Low erucic acid rapeseed oil induced less severe cardiopathologic changes but the nature of the alterations was similar to that of high erucic acid rapeseed oil. Presence of abnormal mitochondria in the cardiac muscle cells of the rats containing focal myocardial necrosis implies that transitions in mitochondrial structure, composition, and function may be responsible for the development of the focal myocardial lesions in rapeseed oil-fed rats.