M. Margaret King

Specificity of a phenobarbital-induced cytochrome P-450 for metabolism of carbon tetrachloride to the trichloromethyl radical

Evidence is presented which demonstrates that the first polypeptide to disappear in liver microsomes of phenobarbital-induced rats treated with CC14 was the 52,000 dalton p-450 cytochrome. Data are also presented which show that this form of cytochrome P-450 was capable of generating the trichloromethyl radical from CCl4 in a reconstituted system containing the purified cytochrome, NADPH-cytochrome P-450 reductase, NADPH, CCl4, and the spin-trapping agent, phenyl-t-butyl nitrone. Other cytochrome P-450 fractions not containing the 52,000 dalton form did not produce this radical. The formation of this highly reactive radical may have resulted in localized damage to the cytochrome, causing the cytochrome either to be released from the microsomal membrane or to form large aggregates which did not migrate in the gel electrophoretic procedures employed.

Comparison of the effect of butylated hydroxytoluene on N-nitrosomethylurea and 7,12-dimethylbenz[a]anthracene-induced mammary tumors

The antioxidant butylated hydroxytoluene (BHT) when fed at a level of 0.3% in a defined semi-purified diet was found to decrease mammary tumor incidence in female Sprague-Dawley rats induced by 7,12-dimethylbenz[a]-anthracene (DMBA). however, no effect of BHT on tumor incidence was seen in animals consuming the same diet, under identical experimental conditions, but treated with the carcinogen nitrosomethylurea (NMU). Differences in effectiveness of BHT as a tumor inhibitor in the 2 model systems, and thoughts as to a possible mechanism of action with regard to BHT are discussed.

A comparison of the efficacy for antitumor activity of the non-steroidal antiestrogens analog II and tamoxifen in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors

Abstract Analog II is a cyclopropyl derivative of stilbene which has been shown to produce antiestrogenic activity with no latent estrogenic effects in the mouse and to reduce the growth of 7,12-dimethylbenz[ a ]anthracene (DMBA)-induced tumors in the rat. Thus, it was of interest to compare the antitumor activity of analog II to the antiestrogen, tamoxifen, which is a partial estrogen agonist in the rat and fully estrogenic in the mouse. The results indicate that analog II and tamoxifen were equally effective in reducing the growth of established DMBA-induced rat mammary tumors and that analog II produced a greater reduction in the occurrence of new tumors during the 7-week treatment period.

Studies on liver microsomes of female rats fed purified diets varying in fat content and with and without propyl gallate

Abstract This work was undertaken to determine whether or not propyl gallate, added as a 0·3% (w/w) supplement to purified diets containing various types and amounts of fat, modifies the function of the hepatic microsomal mixed-function oxidase system. The criteria used included measurements of liver weight, liver to body-weight ratio and hepatic microsomal-protein level as well as analyses of specific components and functions, such as total microsomal cytochrome P -450 content, and the levels of activity of aniline hydroxylase, aminopyrine N -demethylase and NADPH-cytochrome c ( P -450) reductase. In contrast to butylated hydroxytoluene, propyl gallate at a level of 0·3% in the diet did not alter the function of the mixed-function oxidase system, even when administered in a diet high in polyunsaturated fat, which has a permissive effect on the induction of hepatic cytochrome P -450 levels by phenobarbital and 3-methylcholanthrene.

Methods of extraction and high-performance liquid chromatographic analysis of butylated hydroxytoluene from the tissues and serum of rats

Butylated hydroxytoluene (BHT) is a phenolic antioxidant which is widely used in foods and has been shown to inhibit chemical carcinogenesis in the mammary gland induced by 7,12-dimethylbenz(a)anthracene. However, its mechanism of action as a tumor inhibitor is unclear. The purpose of this work was first to develop a method for extracting and quantitating BHT and then to determine the amounts that accumulate in the tissues and serum of rats as a starting point for looking at mechanistic possibilities in the inhibition of mammary carcinogenesis. Methodology of extracting BHT from rat tissues and serum was developed using a modified lipid extraction procedure. The sensitive nature of reverse-phase high-performance liquid chromatography proved useful in detecting and quantifying BHT after its extraction from biological tissues. All tissues were taken from animals consuming semipurified diets with and without 0.3% BHT for various periods of time (weeks). BHT was found in much higher levels in mammary tissue than in the liver and serum of rats. The lipid content in mammary tissue appears to be predictive of the amount of BHT found in this tissue, presumably because of the lipophilic character of the antioxidant.

Effects of butylated hydroxytoluene and acetylaminofluorene on NADPH-cytochrome P-450 reductase activity in rat liver microsomes

Cytochrome P-450 content and NADPH-cytochrome P-450 reductase activity were measured in liver microsomes prepared from male weanling rats fed low-fat, high-saturated fat or high-polyunsaturated fat diets with or without butylated hydroxytoluene (BHT) and 2-acetylaminofluorene (AAF). The inclusion of BHT and/or AAF in the diets consistently produced marked decreases in cytochrome P-450 reductase activity, regardless of the amount and type of dietary fat. In contrast, there was no inhibition of reductase activity when the compounds were added in vitro to liver microsomes.

Possibilities for Dietary Fat and antioxidants as Modulators of Mammary Carcinogenesis

The problem of defining a threshold for even a single carcinogen is both old and new. If one defines “threshold” as the quantity of carcinogen or other etiologic agent just capable of producing a tumor, then at least a common ground is described. However, this “threshold level” may be modulated upwards or downwards in the organism by various environmental and internal factors. A specific case in point is the known influence of dietary fat, especially polyunsaturated fat as an enhancer of many types of tumors, i.e., it appears to be acting to lower the threshold level of carcinogen that is required to elicit a tumorigenic response. Conversely, many dietary antioxidants such as BHT, BHA, and propyl galIate have been shown to effectively raise the threshold level, or decrease the tumorigenic response to a given level of carcinogen. The possible mechanisms through which the dietary factors modify tumorigenesis will be discussed. These will include specific influences and interactions of dietary factors on 7, 12-dimethylbenz(a) anthracene-induced mammary car-cinogenesis, carcinogen uptake and retention by the target tissue, as well as influences on glandular development which may result in an alteration of the number of possible “targets” available at the time of carcinogen exposure.