Inheritance of resistance to promotion of preneoplastic liver lesions in Copenhagen rats.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7- (GST 7-7) positive liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. The objective of this study was to determine if resistance is inherited in a dominant or recessive manner and to derive an estimate of the number of genetic loci involved. We crossed male and female Cop rats with F344 rats to produce F1 offspring. Backcross rats were generated using female F1 rats and either Cop or F344 males, resulting in B1c and B1f generations, respectively. The male rats from all these crosses were initiated with diethylnitrosamine (200 mg/kg) at 7 to 8 weeks of age and were promoted 3 weeks later with the RH protocol (2-acetylaminofluorene and a two-thirds partial hepatectomy). The rats were sacrificed 3 weeks after the partial hepatectomy and their livers were sectioned and stained for GST 7-7-positive lesions. The susceptibility of F1 rats was in between Cop and F344 rats, having 21.7% +/- 2.0% (mean +/- SEM) of their liver volume occupied by lesions versus 4.2% +/- 0.8% for Cop and 53.0% +/- 5.8% for F344 rats. As expected, B1c rats had a volume of liver occupied by lesions that was in between the F1 and Cop rats at 13.5% +/- 1.6%. Surprisingly, B1f rats were similar to B1c rats in their resistance (9.1% +/- 2.1%). These results point to a complex, polygenic inheritance pattern that can be explained by a minimum of four loci, one of which shows recessive epistasis.

Modulatory effects of neonatal exposure to TCDD, or a mixture of PCBs, p,p'-DDT, and p-p'-DDE, on methylnitrosourea-induced mammary tumor development in the rat.

The role of organochlorine (OC) exposure in the etiology of breast cancer remains controversial. Thus, our objective was to determine whether the most abundant and toxic OCs found in human milk could, when ingested during the neonatal period, modulate the development of mammary tumors in the rat. We prepared a mixture composed of p,p'-dichlorodiphenyltrichloroethane (DDT), its major metabolite, p,p'-dichlorodiphenyldichloroethene (DDE), and 19 polychlorinated biphenyls (PCB) based on their concentrations found in the milk of Canadian women. Neonate rats at 1, 5, 10, 15, and 20 days of age were gavaged with this mixture, at 10, 100, and 1,000 times the amount that a human baby would consume. An additional group received 2.5 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg body weight (bw) by gavage at 18 days of age, instead of the mixture. On day 21, all treatment groups, except for a control group and a 1,000-mix group, received a single intraperitoneal injection of methylnitrosourea (MNU, 30 mg/kg bw), the initiator of the carcinogenic process. The average number of rats per treatment group was 33. Rats were sacrificed when their tumors reached 1 cm in size, or at 308 days of age. We prepared mammary tumors and mammary gland whole mounts for histologic analysis. There were no significant effects when only the malignant or only the benign tumors were considered. After all benign and malignant lesions were pooled, the number of mammary tumors differed among all MNU-treated groups (p = 0.02) with more lesions developing in the MNU-1,000[times] (median = 4.5; p = 0.05) and MNU-TCDD (median = 5.5; p = 0.07) compared to the MNU-0 rats (median = 2). Compared to the MNU-0 group, the percentage of rats that developed palpable tumors (benign plus malignant) was slightly higher (p = 0.06) in the MNU-TCDD group, but not in the MNU-1,000[times] group. The percentage of palpable tumors that were malignant was higher (p = 0.02) in the MNU-100[times] group (15/16, 94%) than in the MNU-0 group (10/18, 56%). The highest dose of the mixture delayed (p = 0.03) the development of tumors, but this was not observed with the MNU-TCDD treatment. These results suggest that neonatal exposure to high doses of organochlorines could favor the development of MNU-induced mammary lesions, but also delays the development of palpable tumors in the rat.

Matrix metalloproteinases-2 and 9 do not play a role in the growth of preneoplastic liver lesions in F344 rats.

Matrix metalloproteinases- (MMPs) 2 and 9 (gelatinases A and B) have been implicated in tumor invasion and metastasis, and recent studies have shown increased levels of these enzymes during recovery from partial hepatectomy (PH) in rats. F344 rats are highly susceptible to the growth of glutathione S-transferase 7-7- (GST 7-7) positive preneoplastic liver lesions promoted using the modified resistant hepatocyte (RH) protocol. Since the RH protocol consists of 2-acetylaminofluorene (2-AAF) followed by a PH, we reasoned that MMP-2 and -9 might be critical for the growth of lesions. Using gelatin zymography, we examined the expression of these enzymes in the livers of F344 rats treated with the RH protocol and sacrificed on Days 2, 4, 7, 14, and 21 after 2-AAF/PH. We found increases in both pro- and active MMP-2 and -9 over baseline levels, with the highest levels occurring on Day 7 post-PH. Also, a 54-kDa band, likely to be proMMP-1, was elevated in a pattern similar to MMP-2 and -9. In contrast to F344 rats, identically treated Copenhagen rats that are highly resistant to promotion of liver lesion growth using the RH protocol had significantly lower levels of proMMP-1 and -2. To test the importance of these MMPs to the growth of liver lesions, F344 rats that had been initiated with diethylnitrosamine were treated using the RH protocol. They then received either the MMP inhibitor batimastat (30 mg/kg, intraperitoneally) or vehicle alone daily from Day 3 to 20 post-PH and were sacrificed on Day 21. There were no differences in the percentage of liver volume occupied by GST 7-7-positive lesions (19.1 +/- 4.84 vs 19.4 +/- 3.31, treated versus vehicle, mean +/- SEM) or liver weight as a percentage of body weight (4.11% +/- 0.15 vs 4.07% +/- 0.18, treated versus vehicle, mean +/- SEM) between the treated and control groups. Treatment of rats with batimastat clearly did not affect lesion growth or liver regeneration following the RH protocol. These results suggest that increases in gelatinase expression during the RH protocol are a result of the promotional stimulus rather than a mechanism by which 2-AAF/PH causes lesion growth.

Genetic aspects of nutrition and toxicology: report of a workshop.

The health and resilience of humans and animals is, in large part, determined by the quality and quantity of the diet. This, in turn, may influence an individual's capability to deal with stress including toxic insult. In addition, there may be specific components of the diet that modulate the toxicity of specific toxicants whether the latter are ingested as food or absorbed via other routes. Many examples attest to the importance of interactions between dietary components and toxicants after absorption in the body. Such interactions occur at every level of biological organization from the molecular to the whole organism. Some may be synergistic, others antagonistic. Some may involve direct chemical reaction between the nutrient molecule and the toxicant, others may occur by indirect action at the cellular or organ levels. All examples point to the importance of considering diet when measuring the response to toxic agents whether in animals or humans. In order to foster interaction between the sciences of nutrition and toxicology, The Heinz Institute of Nutritional Sciences as sponsoring a series of workshops. The first of these was held in June, 1999 at the University of Ulster to address evolutionary aspects of nutrition--toxicology (for report see Eur. J. Nutr, 39, 49-52, 2000). In June, 2000, a second workshop was held at the University of Toronto to address genetic aspects, and this is a brief summary of the proceedings. We are beginning to understand the molecular basis of the regulation of gene expression by dietary factors and how genetic changes can affect response to toxicants. Recent advances in technology and a detailed understanding of disease etiology has led to the ability to study molecular determinants of disease risk. The workshop provided a forum for nutritionists, toxicologists, molecular biologists, epidemiologists and others to discuss common interests and to merge their efforts towards an integrated approach to nutrition--toxicology via genetics and genomics. The first session dealt with the mechanism by which nutrients such as fatty acids (Clarke), amino acids (Jefferson) and metal ions (Cousins) can regulate gene expression. In the second session, there were presentations on the effects of nutritional factors on genes of toxicological significance such as phase I and phase II enzymes of drug metabolism (Guengerich, Goodfellow and Grant) as well as on oxidative DNA damage and its repair (Collins, Weindruch). Session three dealt with gene-nutrient interactions in the development of chronic diseases such as diabetes (Hegele, Berdanier) and cancer (Kim, Ambrosone et al.). New developments such as DNA microarrays (McGlynn) and the use of transgenic and knockout models (Sehayek) were presented in the final session.

Resistance of Copenhagen rats to hepatocarcinogenesis does not involve T-cell immunity.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant, compared with susceptible F344 rats, to the growth of glutathione S-transferase 7-7 (GST 7-7) positive preneoplastic liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. Donryu rats, a strain with a level of susceptibility similar to F344, have a reduced T-cell response compared with the closely related, but highly resistant, DRH rat. Cop and DRH rats share several characteristics in their resistance to preneoplastic liver lesion growth and this study, therefore, was designed to examine whether T-cells play a role in Cop resistance. Cop rats were crossed with an athymic (nude) rat to produce F1s that were then interbred to produce F2 animals, some of which were nude with a partial Cop background. A comparison of the susceptibility of nude F2 animals and their euthymic (non-nude) littermates allowed us to determine what role, if any, T-cells play in Cop resistance. We treated 11 Cop, 11 F344, 19 nude F2s, and 18 non-nude F2s with diethylnitrosamine (DEN), followed 3 weeks later by a modified RH protocol. As expected, F344 rats were highly susceptible, having 41.9 +/- 3.3% (mean +/- SEM) of their liver section areas occupied by GST 7-7-positive lesions and Cop rats were highly resistant, having only 4.7 +/- 1.1% of their liver section areas occupied by lesions. Both nude and non-nude F2s were, like Cop rats, highly resistant (1.8 +/- 0.29 and 2.7 +/- 0.45%, respectively). These results show that T-cells are unnecessary for Cop rat resistance, or only play a minor role, and that the nude parental strain is also likely to be resistant to the growth of preneoplastic liver lesions.

Susceptibility of lean and obese Zucker rats to tumorigenesis induced by N-methyl-N-nitrosourea.

To address the possible involvement of hyperinsulinemia in breast cancer development, we have examined the susceptibility of lean and obese Zucker rats to N-methyl-N-nitrosourea (MNU)-induced mammary cancer. Fifty-day-old female lean or obese Zucker rats received intraperitoneal (i.p.) injections of 37.5 or 20 mg/kg MNU, respectively. We showed in separate experiments that these doses produce similar levels of DNA methylation in the mammary epithelial cells of the lean and obese animals. Over the course of 29 weeks following MNU treatment, half of the lean rats developed carcinomas of the mammary gland, demonstrating that they are of intermediate susceptibility to mammary tumorigenesis. During this period, the obese rats developed hyperinsulinemia and insulin resistance as expected. Although palpable tumors developed at a similar rate in the lean and obese rats, only 10% of the obese animals developed mammary carcinomas. The obese rats, however, developed a high incidence (63.3%) of epidermal cysts that occurred mainly in the region of the mammary glands. A 13.3% incidence of colon carcinomas was also found in the obese rats. These results suggest that the development of hyperinsulinemia does not render the obese Zucker rats more susceptible to mammary gland carcinogenesis. Our observation of colon carcinomas in obese, but not lean rats, however, is consistent with evidence that hyperinsulinemia promotes colon cancer in rodents and humans.

Conjugated linoleic acid does not inhibit development of aberrant crypt foci in colons of male Sprague-Dawley rats.

Conjugated linoleic acid (CLA) is a potent inhibitor of the initiation and promotion of mammary carcinogenesis in animal models, but its role in colon carcinogenesis remains unclear. The objective of this study was to determine whether CLA inhibits the promotion of colon carcinogenesis. Forty male Sprague-Dawley rats were given a single dose of azoxymethane (20 mg/kg body wt i.p.). After 1 wk, the animals were randomized into two groups (n = 20) and fed a control AIN-93G diet or the control diet supplemented with 1% CLA at the expense of the soybean oil. After 12 wk, the animals were killed, and their colons were stained with methylene blue for aberrant crypt foci (ACF) analysis by light microscopy. The total number of ACF per animal did not differ between the control (174 +/- 11) and CLA (170 +/- 10) groups. Furthermore, CLA did not affect the average crypt multiplicity (crypts/ACF) or the average number of ACF in any size category. However, rats fed the 1% CLA diet had significantly higher serum insulin levels at the time of sacrifice than those fed the control diet. Thus it is possible that the promoting effects of elevated serum insulin on colon carcinogenesis may have counteracted an inhibitory effect of CLA.

Induction of hepatic insulin-like growth factor binding protein-1 (IGFBP-1) in rats by dietary n-6 polyunsaturated fatty acids.

The insulin-like growth factors (IGFs) are mitogenic polypeptides that have been linked to a variety of normal physiological processes as well as neoplasia. Overexpression of several components of the IGF system is associated with hepatocarcinogenesis in humans and rodents. In rat liver, diets rich in n-6 polyunsaturated fatty acid (PUFA) enhance the development of preneoplastic lesions and tumors. Therefore, the objective of this study was to determine the effect of these dietary fatty acids on the hepatic expression of the various components of the IGF system. The mRNA levels of IGF-1 and the type 1 receptor were not different in livers of rats fed a diet containing 20% corn oil (CO) compared with those fed 5% CO. Analysis of the IGF binding proteins revealed that insulin-like growth factor binding protein-1 (IGFBP-1) levels were altered by the amount and type of dietary fat. A 2.5-fold induction of IGFBP-1 mRNA occurred within 1 week after the animals were fed the 20% corn oil diet compared with those fed 5% CO and was further enhanced to over 6-fold after 1 month. Furthermore, IGFBP-1 protein was only detectable in the livers of animals fed the 20% CO diet. Induction of IGFBP-1 mRNA (4.5-fold) also occurred in rats fed a high-fat diet containing safflower (rich in n-6 PUFAs) compared with those fed a high-fat diet containing menhaden oil (rich in n-3 PUFAs). The induction of IGFBP-1 mRNA was independent of serum insulin levels and the development of insulin resistance. Since IGFBP-1 mRNA is upregulated in regenerating liver, we reasoned that the induction of IGFBP-1 mRNA may be associated with an increase in cell proliferation; however, no difference was observed in the hepatic labeling index of rats fed the 20% CO compared with the 5% CO diet. In summary, these studies show a striking induction by dietary n-6 PUFAs of hepatic IGFBP-1, a protein that has been implicated in liver cancer development.

Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations.

Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene.

Regulation of mevalonate synthesis in low density lipoprotein receptor knockout mice fed n-3 or n-6 polyunsaturated fatty acids.

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, catalyzes the formation of mevalonate which is also required for cell proliferation. Changes in HMG-CoA reductase may mediate the differential effects of n-3 and n-6 polyunsaturated fatty acids (PUFA) on experimental mammary tumorigenesis, but the mechanisms by which these fatty acids regulate HMG-CoA reductase are unclear. To determine whether the low density lipoprotein receptor (LDL-R) is required for this regulation, groups of female LDL-R knockout (-/-) and wild-type (+/+) mice were fed 7% fat diets rich in either n-3 (menhaden oil) or n-6 (safflower oil) PUFA for 1 wk. Dietary PUFA and deletion of the LDL-R had independent effects on HMG-CoA reductase and serum lipids, and a significant diet-gene interaction was observed. The effects of PUFA on HMG-CoA reductase in the mammary gland, but not the liver, were mediated by the LDL-R. We also observed that differences in HMG-CoA reductase and serum LDL-cholesterol, high density lipoprotein cholesterol, and triglycerides between -/- and +/+ mice were dependent on whether the mice were fed n-3 or n-6 PUFA. Differences between -/- and +/+ mice were much greater when animals were fed n-6 PUFA rather than n-3 PUFA. These results show that the LDL-R mediates the effects of PUFA on HMG-CoA reductase in the mammary gland but not the liver. Furthermore, the composition of dietary PUFA profoundly influences the effects of deleting the LDL-R on HMG-CoA reductase and serum lipids and suggests that diet may influence the phenotype of other knock-out or transgenic animals.

Resistance to the promotion of glutathione S-transferase 7-7-positive liver lesions in Copenhagen rats.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7 (GST 7-7)-positive liver lesions following treatment with a modified resistant hepatocyte protocol. The objective of the current study was to establish the time course for the development of resistance and examine potential resistance mechanisms in Cop rats using F344 rats as susceptible controls. Male Cop and F344 rats (n = 25), 7-8 weeks of age, were initiated with diethylnitrosamine (200 mg/kg) and promoted 3 weeks later with four doses of 2-acetylaminofluorene (20 mg/kg) and a 2/3 partial hepatectomy (PH). Groups of rats from each strain were killed on days 2, 4, 7, 14 and 21 post-PH, 2 h after receiving bromodeoxyuridine. Cop livers contained similar numbers of GST 7-7-positive lesions to F344 livers on days 2 and 4 post-PH. The percent volume of liver occupied by these lesions did not differ between the strains on days 2, 4 and 7 post-PH. On day 14, however, approximately 29% of the liver volume in F344 rats was occupied by lesions, whereas in Cop rats this was significantly less (approximately 9%, P < 0.001). On day 21, lesions occupied approximately 58% of F344 rat livers and only approximately 6% of Cop livers. Despite these differences, the labeling index of hepatocytes was not significantly different between the strains at any time point, either within lesions or within surrounding normal liver. Furthermore, the apoptotic indices were not different between the strains at any time. However, differences were found in the extent of lesion remodeling (redifferentiation) and in the pattern of oval cell response following PH in Cop livers. By day 14 post-PH, approximately 76% of Cop liver lesions showed evidence of remodeling, compared with only approximately 14% of F344 lesions. The oval cell response to PH was equivalent in the two strains up to day 4 post-PH but by day 7, in F344 livers there was extensive migration of these cells into the liver parenchyma, whereas in Cop livers, the response remained localized to the portal regions. These results suggest that Cop resistance occurs at the promotion stage and not the initiation stage of carcinogenesis. Resistance appears not to be due to a lower proliferation rate nor to a higher apoptotic rate within Cop lesions. Precocious remodeling and/or a diminished oval cell response, however, may contribute to the resistance of Cop rats to the growth of GST 7-7-positive hepatic lesions.

Resistance to mammary tumorigenesis in Copenhagen rats is associated with the loss of preneoplastic lesions.

The resistance of Copenhagen (Cop) rats to mammary tumor development has recently been linked to three loci, but the genes have yet to be cloned and the mechanism of resistance is still largely unknown. In order to determine the cellular events associated with resistance, we prepared mammary whole mounts from Cop and susceptible Wistar Furth (WF) rats 0, 15, 30, 45 and 60 days after treatment with 50 mg/kg N-methyl-N-nitrosourea (MNU). At 15 days, treated rats of both strains had significantly more undifferentiated structures [terminal end buds (TEBs)] and significantly fewer differentiated structures [alveolar buds (ABs)] than untreated rats. Treated Cop rats, however, had significantly more TEBs and fewer ABs than age-matched, treated WF rats. Histological analysis of preneoplastic lesions tentatively identified from the whole mounts showed that like WF rats, Cop rats developed early preneoplastic lesions [intraductal proliferations (IDPs)] by 15 days post-MNU treatment. Unlike IDPs from WF rats, however, the IDPs in Cop rats then decreased in number until they were absent 60 days post-MNU treatment. Furthermore, they failed to progress into more advanced lesions such as ductal carcinomas in situ (DCIS). Finally, we found G-->A activating mutations in codon 12 of the Ha-ras gene in 60% of IDPs from Cop rats and 75% of IDPs from WF rats. Our results show that resistance in Cop rats is not due to a target cell population for the carcinogen that is smaller than in susceptible rats or to the failure of the carcinogen to inhibit mammary gland differentiation. Furthermore, we have shown that Cop rats develop preneoplastic IDPs that harbor Ha-ras mutations but, unlike IDPs in susceptible strains, they fail to progress and ultimately disappear.

Cyclin D1 expression during rat mammary tumor development and its potential role in the resistance of the Copenhagen rat.

BACKGROUND: Resistance to mammary tumorigenesis in Copenhagen rats is associated with loss of early preneoplastic lesions known as intraductal proliferations. The cause of this disappearance, however, is unknown. RESULTS: There were no differences in the numbers of lesions in mammary whole-mounts prepared from Copenhagen or Wistar-Furth rats at 20 or 30 days after N-methyl-N-nitrosourea treatment, but at 37 days there were significantly fewer lesions in Copenhagen glands. Furthermore, lesions in Copenhagen glands were exclusively intraductal proliferations, whereas in Wistar-Furth glands more advanced lesions were also present. Immunohistochemical staining showed frequent cyclin D1 overexpression in Wistar-Furth lesions at 37 days, but not in Copenhagen lesions. There were, however, no differences in p16INK4a protein expression, bromodeoxyuridine labeling and apoptotic indices, or mast cell infiltration between Copenhagen and Wistar-Furth lesions at any time. CONCLUSIONS: Overexpression of cyclin D1 in preneoplastic lesions may be important in the development of mammary tumors in susceptible rats, although this overexpression does not appear to cause significant changes in cell kinetics. Furthermore, the low levels of cyclin D1 expression in Copenhagen intraductal proliferations may play a role in the resistance of these rats to mammary tumorigenesis.

Effect of hormonal status on the expression of the cyclooxygenase 1 and 2 genes and prostaglandin synthesis in rat mammary glands.

Hormonal effects on mammary carcinogenesis have been linked to prostaglandin (PG) synthesis. The purpose of the present study was to examine the expression of the cyclooxygenase (COX) 1 and 2 genes and levels of PG synthesis in the mammary glands of rats that have different levels of susceptibility to mammary gland carcinogenesis associated with pregnancy, lactation, post-lactation involution, and ovariectomy. The expression of COX-1 mRNA, measured by Northern blot analysis, was similar in virgin, lactating, pregnant, and post-lactational animals of the same age. Ovariectomized animals exhibited significantly lower levels of COX-1 mRNA (approximately 40%) compared to the sham-operated controls or the ovariectomized animals treated with estradiol and progesterone. COX-2 mRNA, measured by RT-PCR, was detectable only in the mammary glands of lactating animals and ovariectomized animals administered estradiol and progesterone. Induction on COX-2 expression occurred in both stromal and epithelial cells in lactating rat mammary glands. COX enzymatic activities, determined by measuring the conversion rate of [1-14 C]-arachadonic acid to prostanoids, showed that lactating animals had a significantly higher activity compared to virgin (approximately 40%), pregnant (approximately 30%), or postlactational animals (approximately 40%). Ovariectomized animals had significantly lower COX enzymatic activity compared to the sham operated animals. Significant induction of COX activity, however, was observed in ovariectomized animals administered estradiol and progesterone. These changes in COX enzymatic activity were paralleled by similar changes in the mammary gland PGE2 content, measured by enzyme immunoassay. Our results suggest that the effect of hormones on the genesis of mammary cancer in the rat may be mediated, at least in part, by their effects on COX-2 expression and PG synthesis.

Zonal location of compensatory hepatocyte proliferation following chemically induced hepatotoxicity in rats and humans.

Hepatocyte proliferation stimulated by partial hepatectomy occurs first in periportal cells, with midlobular and then perivenous cell division occurring later. We have previously shown that this pattern of compensatory cell proliferation also occurs following the hepatotoxicity of N-nitrosodimethylamine. We examined the generality of this pattern in livers of rats given a minimally toxic dose of an hepatotoxin and in liver biopsy samples from patients who had taken overdoses of acetaminophen. Proliferating hepatocytes were detected immunohistochemically (5'-bromodeoxyuridine or Ki-67 antigens). The perivenous necrogens N-nitrosodiethylamine, carbon tetrachloride (CCl4), bromobenzene, and acetaminophen all induced periportal hepatocyte proliferation. With CCl4, bromobenzene, and acetaminophen, the initial appearance of proliferating periportal hepatocytes was followed 12-24 hr later by division in the midlobular region, with a few cells dividing adjacent to the perivenous region of necrosis. The periportal necrogen allyl alcohol also induced periportal cell division. In the human studies, perivenous necrosis was accompanied by periportal and midlobular hepatocyte proliferation. These results suggest that regardless of its lobular location chemically induced hepatotoxicity stimulates cell proliferation that begins in the periportal zone and then moves in an orchestrated response into the midlobular and perivenous zones.

The effect of dietary n-3 and n-6 polyunsaturated fatty acids on the expression of cyclooxygenase 1 and 2 and levels of p21ras in rat mammary glands.

Dietary n-6 polyunsaturated fatty acids (PUFAs) promote rat mammary cancer while n-3 PUFAs are inhibitory. The purpose of this study was to determine whether the fats exert their effects by altering the expression of genes that affect cancer development. Therefore, we have examined the effect of PUFAs on the expression of the cyclooxygenase (COX) 1 and 2 genes that are involved in prostaglandin biosynthesis. We also investigated the effect of dietary PUFAs on the expression of the p21ras protein and Ha-ras mRNA. Rats were fed either low- (7%; LF) or high- (21%; HF) fat diets that were rich in either n-6 PUFAs (safflower oil, S) or n-3 PUFAs (menhaden oil, M) for 3 weeks. COX-1 mRNA levels were approximately the same in groups fed diets containing either level of menhaden oil, but were increased by approximately 30% in the LFS and HFS groups (P < 0.05). Transcripts of the inducible COX-2 gene were not detectable in the menhaden oil groups, but this gene was expressed in animals fed either level of safflower oil and in the HFS group was associated with increased levels of COX enzymatic activity and production of PGE2. Animals fed safflower oil had elevated levels of p21ras protein compared to animals fed menhaden oil. Ha-ras mRNA was increased by approximately 35% in animals fed HFS compared to the group fed HFM (P < 0.05). These results demonstrate that dietary n-6 PUFAs upregulate COX-2 and, to some extent, COX-1 expression. There was a concomitant increase in COX enzyme activity and PG synthesis in the mammary glands of rats fed high levels of n-6 PUFAs. Together with associated changes in p21ras expression, these results may explain, at least in part, the promoting effects of dietary n-6 PUFAs on mammary carcinogenesis.