Kelly E. Patrick

Eicosapentaenoic and arachidonic acid: comparison of metabolism and activity in murine epidermal cells.

The biological activity, including metabolism and modulation of ornithine decarboxylase activity and DNA synthesis, of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were compared in epidermal cells from SENCAR mice. Radiolabelled AA and EPA were found to be similarly incorporated into and released from membrane phospholipids of unstimulated cultures. However, when cells were stimulated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), the release of AA was significantly higher than the release of EPA. The extent of metabolism of AA and EPA to prostaglandins was determined in both freeze-thawed cell preparations and in viable cultured cells. In the freeze-thawed preparations, use of AA as a substrate resulted in significantly more PGF than when EPA was used as the substrate. However, more PGE3 was formed than PGE2. PGD levels were the same for either fatty acid precursor. Prostaglandin production was also determined in viable cultured cells since other influences such as phospholipase A2 activity can modify prostaglandin production. Control cultures prelabelled with either AA or EPA produced similar amounts of the respective PGF, PGE, and PGD. However, TPA-stimulated cultures produced significantly higher amounts of each prostaglandin in cultures prelabelled with AA compared to cells prelabelled with EPA. HETE or HEPE production was the same both for cultured cells prelabelled with AA or EPA and for homogenates from uncultured cells incubated directly with the radiolabelled fatty acids. TPA-induced ornithine decarboxylase (ODC) was significantly higher in AA-treated cultures compared to EPA-treated cultures. AA supports DNA synthesis to a greater extent than EPA, either alone or in the presence of TPA. These findings suggest that AA and EPA do not have equivalent biological activity in mouse epidermal cells.

The effect of the level of dietary corn oil on mouse skin carcinogenesis

To investigate the effects of two levels of dietary corn oil on tumorigenesis, semipurified diets containing 5% or 10% corn oil were fed during the promotion stage of a mouse skin carcinogenesis model. Sencar mice were initiated with 10 nmol dimethylbenz[a]anthracene (DMBA) and promoted with either 1 microgram 12-O-tetradecanoylphorbol-13-acetate (TPA) or 40 mg benzoyl peroxide twice weekly for 24 or 52 weeks, respectively. No significant differences in kilocalories of food consumed or body weights were observed between the diet groups during the study. Fatty acid profiles of the epidermal phospholipids reflected dietary fat intake. For example, high levels of linoleate and low levels of arachidonate were found in the phosphatidylcholine fraction from mice fed the 10% corn oil diet compared with 5% corn oil. When the diets were fed during TPA promotion, the papilloma incidence after 11 weeks of treatment for the 5% corn oil group was 77% and 37% for the 10% corn oil group. By 15 weeks of TPA treatment, papilloma incidence between the diet groups was similar, and later, carcinoma incidence and yield were not different between the two groups. For the animals treated with benzoyl peroxide, there was only a slight but not significant difference in papilloma and carcinoma appearance. In parallel studies, ornithine decarboxylase activity, vascular permeability, hyperplasia, and prostaglandin E2 (PGE2) levels were elevated in the epidermis after promoter treatment, but only hyperplasia and PGE2 synthesis tended to reflect the dietary effects on tumor appearance. These data suggest that the quantity of dietary corn oil at the two levels tested, 5% and 10%, altered epidermal phospholipid fatty acid composition and PGE2 levels and had modest effects on the modulation of tumorigenesis in this skin model.

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.

The effect of various dietary fats on skin tumor initiation

The type of dietary fat has been shown to modulate the initiation stage of mammary tumorigenesis, with saturated fat fed before and/or during carcinogen treatment resulting in increased tumor incidence. This study was designed to determine whether different types of dietary fat alter the initiation stage of skin carcinogenesis by use of the initiation-promotion mouse skin carcinogenesis model. Sencar mice were divided into three groups and maintained on one of the experimental diets. The AIN-76-based diets consisted of 10% total fat with various types of fat: 8.5% menhaden oil plus 1.5% corn oil, 8.5% coconut oil plus 1.5% corn oil, and 10% corn oil. After three weeks mice were initiated with 10 nmol dimethylbenz[a]anthracene (DMBA). Two weeks later, all mice were switched to a diet containing 5% corn oil. Promotion began four weeks after initiation with twice-weekly application of 1 microgram 12-O-tetradecanoylphorbol-13-acetate and continued for 12 weeks. No statistically significant differences in kilocalories of food consumed or body weights were observed between diet groups during the study. The final papilloma incidence, yield, and size were not significantly different among the diet groups. In a parallel study, [3H]DMBA binding to epidermal DNA showed no dietary differences. Unlike the mammary carcinogenesis model, these data suggest that the type of fat fed during DMBA initiation had minimal effects on this stage of skin carcinogenesis.

Tumour promoter-induced release and metabolism of arachidonic acid: Comparison between mouse and human epidermal cells

Arachidonic acid (AA) release and metabolism to prostaglandins (PG) in response to the complete mouse skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the first-stage promoter A23187 were compared in keratinocytes from normal adult human and two different strains of mouse with different sensitivities to TPA as a tumour promoter. The rate, extent and distribution of incorporated [(14)C]AA among the classes of phospholipids were similar in cultures of either CD-1, SSIN or human keratinocytes. Using prelabelled cultures, the amount of radiolabel released in control cultures was nearly identical for human and both strains of mice. Distinct species differences were observed, however, after TPA treatment. Cultures of human epidermal keratinocytes (HEK) released only half as much label by 6 hr compared with either strain of mouse. In addition, whereas the mouse epidermal keratinocytes (MEK) metabolized AA readily to PGE(2), very little PGE(2) could be detected in the human cultures. The extent of variability between individual human samples (n = 11) in response to 0.1 mug TPA/ml ranged from a 20% increase to a 200% increase in release of label, with a mean increase of 50%, whereas murine cells produced a mean increase of 400%. When MEK and HEK were stimulated with 10(-6) and 10(-5)m-A23187, an increase in the release of arachidonate by 200 and 400%, respectively, was observed for both species. Under these conditions of equal release, equivalent amounts of PGE(2) were produced. To compare further the ability of mouse and human cells to metabolize exogenous AA to PGE(2), freshly isolated, as well as cultured, cells from each species were incubated with [(14)C]arachidonate. Under both conditions, HEK have approximately the same ability as MEK to metabolize AA to PGE(2) (approx. 2% of the arachidonate for both species). The reduced ability of HEK, compared with MEK, to produce PGE(2) is specific to TPA and is due primarily to insufficient substrate, that is, low levels of arachidonic acid release.