P. David Josephy

A comparison of the anticarcinogenic properties of four red wine polyphenols.

BACKGROUND There has been growing interest in the analysis of certain polyphenols in wine, especially flavonoids, trihydroxystilbenes and phenolic acids, stimulated by intense research into their potential benefits to human health. One of their main properties in this regard is their antioxidant activity, which enables them to attenuate the development of atherosclerosis, inflammatory diseases, and cancer. METHODS A two stage CD-1 mouse skin cancer model using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and phorbol 12-myristate 13-acetate (TPA) as promoter was employed to compare the antitumorigenic activities of one polyphenol from each of four different classes: flavanols [(+)-catechin], stilbenes (trans-resveratrol), flavonols (quercetin) and hydroxybenzoic acids (gallic acid). Animals were treated with specific polyphenols at doses ranging from 0 to 25 micromoles (dissolved in 200 microL acetone), twice a week for eighteen weeks. The solution was applied topically to the shaved dorsal region of each animal. The relative potencies of the polyphenols were compared by evaluating the percentage inhibition of tumor formation in individual mice and the number of mice developing one or more tumors with the different dose schedules. RESULTS Probit analysis revealed that quercetin was the most (ED(50)<1 micromole) and gallic acid the least effective (ED(50) 5-10 micromoles). (+)-Catechin and trans-resveratrol were intermediate, with ED(50) values of 5 and 6 micromoles, respectively. CONCLUSION We have shown recently that trans-resveratrol is absorbed much more efficiently than (+)-catechin and quercetin in humans after oral consumption. Taking this and the relative concentrations in red wine into account, together with the present results, we conclude that trans-resveratrol may be the most effective anticancer polyphenol present in red wine as consumed po by healthy human subjects.

Recent advances in the construction of bacterial genotoxicity assays.

Bacterial mutagenicity assays have been widely used in genotoxicology research for two decades. We discuss the development of such assays, especially the Ames test, with particular attention to strain engineering. Genes encoding enzymes of mutagen bioactivation, including N-acetyltransferase, nitroreductase, and cytochrome P450, have been introduced into tester strains. The processing of DNA damage by the bacterial strains has also been modified in several ways, so as to enhance mutagenesis. These efforts have greatly increased the sensitivity of mutation assays and have illuminated the molecular mechanisms of mutagenesis. We also discuss the relationship between bacterial assays and in vivo mutation assays which use transgenic rodents.

Myeloperoxidase Genotype, Fruit and Vegetable Consumption, and Breast Cancer Risk

Myeloperoxidase (MPO), an antimicrobial enzyme in the breast, generates reactive oxygen species (ROS) endogenously. An MPO G463A polymorphism exists in the promoter region, with the variant A allele conferring lower transcription activity than the common G allele. Because oxidative stress may play a role in breast carcinogenesis, we evaluated MPO genotypes in relation to breast cancer risk among 1,011 cases and 1,067 controls from the Long Island Breast Cancer Study Project (1996–1997). We also assessed the potential modifying effects of dietary antioxidants and hormonally related risk factors on these relationships. Women over 20 years with incident breast cancer who were residents of Nassau and Suffolk Counties, NY, were identified as potential cases. Population-based controls were frequency matched by 5-year age groups. Genotyping was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) technology, and suspected breast cancer risk factors and usual dietary intake were assessed during an in-person interview. Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals. Having at least one A allele was associated with an overall 13% reduction in breast cancer risk. When consumption of fruits and vegetables and specific dietary antioxidants were dichotomized at the median, inverse associations with either GA or AA genotypes were most pronounced among women who consumed higher amounts of total fruits and vegetables (odds ratio, 0.75; 95% confidence interval, 0.58–0.97); this association was not noted among the low-consumption group (P for interaction = 0.04). Relationships were strongest among premenopausal women. Results from this first study of MPO genotypes and breast cancer risk indicate that MPO variants, related to reduced generation of ROS, are associated with decreased breast cancer risk, and emphasize the importance of fruit and vegetable consumption in reduction of breast cancer risk.

The molecular toxicology of acetaminophen.

The history of the development of the analgestic drug acetaminophen is reviewed with an emphasis on the characteristics of its overdose toxicity. The P450-catalyzed oxidation of acetaminophen generates a reactive electrophile that binds covalently to proteins. Involvement of specific P450 enzymes in acetaminophen toxicity can be probed by experiments with knock-out mice. The identification of specific target proteins may help to clarify the mechanism of acetaminophen hepatoxocity.The history of the development of the analgestic drug acetaminophen is reviewed with an emphasis on the characteristics of its overdose toxicity. The P450-catalyzed oxidation of acetaminophen generates a reactive electrophile that binds covalently to proteins. Involvement of specific P450 enzymes in acetaminophen toxicity can be probed by experiments with knock-out mice. The identification of specific target proteins may help to clarify the mechanism of acetaminophen hepatoxocity.

“Phase I and Phase II” Drug Metabolism: Terminology that we Should Phase Out?

Richard Tecwyn Williams (1909–1979) was one of the founders of the study of drug metabolism. His monograph “Detoxication Mechanisms: The Metabolism of Drugs and Allied Organic Compounds” (Williams, 1949) helped to establish the study of drug and toxicant metabolism as a scientific discipline. Williams summarized the then-known processes of metabolism of xenobiotics, organizing the subject according to classes of foreign chemicals, rather than according to classes of biotransformations. The second edition of the book (Williams, 1959), was again organized by chemical class. However, in a concluding chapter, Williams introduced the idea of distinguishing between two categories of biotransformation, which he called “Phase I” and “Phase II” reactions. The following excerpt from the book defines this distinction. “In the first chapter it was stated that the reactions of foreign compounds in vivo could be divided into four classes, oxidations, reductions, hydrolyses and syntheses. All the reactions of foreign compounds can, in fact, be discussed broadly in terms of these four classes of reactions. The oxidations include not only the commonly accepted oxidations such as the conversion of C-methyl groups, alcohols and aldehydes to carboxylic acids, but also oxidative O- and N-dealkylations, oxidative deaminations, aromatic hydroxylations, dehydrochlorinations, etc… . It appears that the metabolism of most foreign compounds occurs in two phases. The first phase involves oxidations, reductions or hydrolyses or a combination of any of these three, and for convenience these may be termed “phase I reactions”; the second phase (“phase II reactions”) consists of synthesis, mainly conjugations such as glucuronide, ethereal sulphate, thiocyanate, and hippuric acid formation. In the first phase, biologically active compounds may be inactivated or have

Electron spin resonance spin trapping of thiyl, radicals from the decomposition of thionitrites

Abstract Alkyl thiyl radicals produced by the homolytic decomposition of thionitrites are detected by ESR spin trapping using 5,5-dimethyl-1-Δ-pyrroline-N-oxide (DMPO).

Inter-individual differences in the metabolism of environmental toxicants: cytochrome P450 1A2 as a prototype.

Cytochrome P450 (P450) 1A2 provides an interesting paradigm for inter-individual differences in the metabolism of pro-carcinogens. The enzyme is known to vary 40-fold among individuals and may contribute to cancers caused by heterocyclic amines and other chemicals. Rat and human P450 1A2 are known to be 75% identical and were compared for several catalytic activities. The human enzyme was an order of magnitude more efficient in the N-hydroxylation of two heterocyclic amines. Further, the levels of P450 1A2 expressed in human livers show a 40-fold variation, with some as high as 0.25 nmol P450 1A2 per milligram microsomal protein. Some human liver samples are more active (than those isolated from polychlorinated biphenyl-treated rats) in the activation of heterocyclic amines. A bacterial genotoxicity assay has been developed in which human P450 1A2 and NADPH-P450 reductase are expressed within Escherichia coli and bacterial mutants can be assayed using reversion to lac prototrophy. A random mutagenesis strategy for human P450 1A2 has been developed and used to examine the changes in catalytic activity seen with many single-amino acid substitutions. These results may be of relevance in consideration of genetic polymorphisms. Further, the findings pose a challenge to molecular epidemiology effort in that results with one substrate do not necessarily predict those for others. Some dinitropyrenes are P450 1A2 substrates but others are not. 6-Nitrochrysene can be activated by human P450 1A2 but the (mono) nitropyrenes examined were not; these were oxidized by P450 3A4 instead.

Microenvironmental influences on mutagenesis in mammary epithelial cells.

Tumor progression may be viewed as an evolutionary process at the cellular level. Because blood supply to solid tumors is inadequate, the cancer cells face a hostile microenvironment characterized by hypoxia or anoxia, acidic extracellular pH and nutrient deficiencies. It has been proposed that these factors result in increased levels of spontaneous mutagenesis and thereby contribute to tumor progression. We have examined spontaneous mutagenesis in vitro and in vivo, using previously characterized cell lines (mammary epithelial cells [ME] and mammary fibroblast cells [MFib]) from the mammary gland of the BigBlue™ rat, carrying a transgene construct suitable for the detection of mutations. Cells were exposed in vitro to control conditions, low pH, or to glucose deprivation, under normoxic or hypoxic culture conditions, and were also grown as xenografted tumors in immune‐deficient mice. We examined cell survival and mutant frequency/spectrum at the cII locus. Significant increases in mutant frequency were observed in ME cells exposed to hypoxia alone or in combination with no glucose; the latter condition also resulted in reduced clonogenic survival. Cells grown as xenografts and then recovered and expanded in culture also had elevated frequencies of spontaneous mutations. We observed a shift in the spontaneous mutation spectrum between the ME cells and the MET cells (cultured in vitro or isolated from mouse xenograft tumors). These results support the concept that the tumor microenvironment contributes to tumor progression by enhancing spontaneous mutagenesis, that different cell types from the same organ can respond differently to these stresses and that differences in microenvironment may influence the types of mutations that arise.

The Escherichia coli lacZ reversion mutagenicity assay.

The Escherichia coli lacZ reversion assay, based on the set of episomal lacZ alleles engineered by Miller et al., provides an attractive system for studies of mutagenesis and mutational specificity. Each strain in the lacZ set reverts by a specific base substitution or frameshift event. Revertants are selected by growth on lactose minimal medium. In this review, I describe the development of the assay and its subsequent modifications and improvements. Examples of its application are presented and detailed protocols for the implementation of the assay are given.

Inhibition of benzo[a]pyrene dihydrodiol epoxide mutagenicity by synthetic analogues of ellagic acid

Dibenzo[b, d]pyran-6-one, hydroxylated and methoxylated derivatives of this ring system, and some other analogues of the natural product ellagic acid have been synthesized and examined as inhibitors of benzo[a]pyrene dihydrodiol epoxide (BPDE) mutagenicity in Salmonella typhimurium strain TA100. Some of these new compounds have inhibitory effectiveness comparable to the natural product. On the basis of our results, we suggest qualitative rules for predicting inhibitory activity of ellagic acid analogues.