Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases.

The human diet contains a great variety of natural mutagens and carcinogens, as well as many natural antimutagens and anticarcinogens. Many of these mutagens and carcinogens may act through the generation of oxygen radicals. Oxygen radicals may also play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation (and promotion), that may be related to cancer, heart disease, and aging. Dietary intake of natural antioxidants could be an important aspect of the body's defense mechanism against these agents. Many antioxidants are being identified as anticarcinogens. Characterizing and optimizing such defense systems may be an important part of a strategy of minimizing cancer and other age-related diseases.

Identifying environmental chemicals causing mutations and cancer.

Damage to DNA appears to be the major cause of most cancer and genetic birth defects and may contribute to aging and heart disease as well. The agents that cause this damage must be identified. Many of these agents are natural chemicals present in the human diet as complex mixtures. The tens of thousands of man-made chemicals that have been introduced into the environment in the last few decades must also be tested for their ability to damage DNA. Existing animal tests and human epidemiology alone are inadequate for this task because of time, expense, and the difficulty of dealing with complex mixtures, Newly developed short-term tests, most of them assaying for mutagenicity, are discussed as key tools in identifying environmental mutagens and carcinogens.

Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation.

The oxyR gene positively regulates genes induced by oxidative stress in Salmonella typhimurium and Escherichia coli. Purification of the OxyR protein showed that oxidized but not reduced OxyR activates transcription of oxidative stress-inducible genes in vitro. Conversion between the two forms of OxyR is rapid and reversible. Both the oxidized and the reduced forms of the OxyR protein are capable of binding to three diverse sequences upstream of OxyR-regulated promoters, but the interactions of the two forms of OxyR with the promoter regions are different. The results suggest that direct oxidation of the OxyR protein brings about a conformational change by which OxyR transduces an oxidative stress signal to RNA polymerase.

Ranking possible carcinogenic hazards.

This review discusses reasons why animal cancer tests cannot be used to predict absolute human risks. Such tests, however, may be used to indicate that some chemicals might be of greater concern than others. Possible hazards to humans from a variety of rodent carcinogens are ranked by an index that relates the potency of each carcinogen in rodents to the exposure in humans. This ranking suggests that carcinogenic hazards from current levels of pesticide residues or water pollution are likely to be of minimal concern relative to the background levels of natural substances, though one cannot say whether these natural exposures are likely to be of major or minor importance.

Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens.

K-region epoxides of the carcinogens benz[a] anthracene, dibenz[a,h]anthracene, and 7-methylbenz[a] anthracene are mutagenic in strains of Salmonella typhimurium designed to detect frameshift mutagens. Parent hydrocarbons, K-region diols and phenols and some other epoxides are inactive as mutagens in these tests. Polycyclic hydrocarbon epoxides, and other presumed proximal carcinogens, are discussed as examples of intercalating agents with reactive side chains. It has been shown previously that intercalating agents with reactive side chains are potent frameshift mutagens.

Another flame retardant, tris-(1,3-dichloro-2-propyl)-phosphate, and its expected metabolites are mutagens.

A flame retardant used in children's sleepwear, tris-(1,3-dichloro-2-propyl)phosphate (Fyrol FR2) is a mutagen in the Salmonella-mammalian tissue homogenate test after it has been activated by mouse or rat liver homogenate. The expected enzymatic hydrolysis product, 1,3-dichloro-2-propanol, is similarly a mutagen after activation by liver homogenate. A proposed metabolite of the flame retardant, 1,3-dichloro-2-propanone, is a potent mutagen in the absence of such activation. A flame retardant with similar structure, tris-(2,3-dibromopropyl)phosphate (tris-BP), was shown previously to be a mutagen, to cause sterility in animals, to be a carcinogen, and to be absorbed through human skin. These and other flame retardants have characteristic nuclear magnetic resonance spectra that can be used to determine which flame retardant is present in commercially purchased sleepwear. Sleepwear treated with tris-BP, Fyrol FR2, and other chemical additives was being sold in late 1977.

Bilirubin is an antioxidant of possible physiological importance.

Bilirubin, the end product of heme catabolism in mammals, is generally regarded as a potentially cytotoxic, lipid-soluble waste product that needs to be excreted. However, it is here that bilirubin, at micromolar concentrations in vitro, efficiently scavenges peroxyl radicals generated chemically in either homogeneous solution or multilamellar liposomes. The antioxidant activity of bilirubin increases as the experimental concentration of oxygen is decreased from 20% (that of normal air) to 2% (physiologically relevant concentration). Furthermore, under 2% oxygen, in liposomes, bilirubin suppresses the oxidation more than alpha-tocopherol, which is regarded as the best antioxidant of lipid peroxidation. The data support the idea of a "beneficial" role for bilirubin as a physiological, chain-breaking antioxidant.

Toxaphene, a complex mixture of polychloroterpenes and a major insecticide, is mutagenic.

Toxaphene, the most widely used chlorinated insecticide, is mutagenic in the Salmonella test without requiring liver homogenate for activity. This insecticide is a complex mixture (more than 177 polychloroterpenes) with carcinogenic activity in rodents. Some but not all of the mutagenic components are easily separated from the insecticidal ingredients.

Rodent carcinogens: setting priorities.

The human diet contains an enormous background of natural chemicals, such as plant pesticides and the products of cooking, that have not been a focus of carcinogenicity testing. A broadened perspective that includes these natural chemicals is necessary. A comparison of possible hazards for 80 daily exposures to rodent carcinogens from a variety of sources is presented, using an index (HERP) that relates human exposure to carcinogenic potency in rodents. A similar ordering would be expected with the use of standard risk assessment methodology for the same human exposure values. Results indicate that, when viewed against the large background of naturally occurring carcinogens in typical portions of common foods, the residues of synthetic pesticides or environmental pollutants rank low. A similar result is obtained in a separate comparison of 32 average daily exposures to natural pesticides and synthetic pesticide residues in the diet. Although the findings do not indicate that these natural dietary carcinogens are important in human cancer, they cast doubt on the relative importance for human cancer of low-dose exposures to synthetic chemicals.

Children absorb tris-BP flame retardant from sleepwear: urine contains the mutagenic metabolite, 2,3-dibromopropanol.

The flame retardant, tris(2,3-dibromopropyl)phosphate (tris-BP), which is a mutagen and causes cancer and sterility in animals is absorbed from fabric by people. 2,3-Dibromopropanol, a metboloite of tris-BP and a mutagen itself, has been found in the urine samples of ten children who were wearing or who had worn tris-BP-treated sleepwear. Eight of these children were wearing well-washed sleepwear and the possibility of absorption of tris-BP from well-washed sleepwear discussed. 2,3-Dibromopropanol was not found in the urines of one child and one adult who had never worn tris-BP-treated garments.

Gamma-tocopherol prevents airway eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma.

BACKGROUND: Traditional therapies for asthma and allergic rhinitis (AR) such as corticosteroids and antihistamines are not without limitations and side effects. The use of complementary and alternative approaches to treat allergic airways disease, including the use of herbal and dietary supplements, is increasing but their efficacy and safety are relatively understudied. Previously, we have demonstrated that gamma-tocopherol (gammaT), the primary form of dietary vitamin E, is more effective than alpha-tocopherol, the primary form found in supplements and tissue, in reducing systemic inflammation induced by non-immunogenic stimuli. OBJECTIVE: We used allergic Brown Norway rats to test the hypothesis that a dietary supplement with gammaT would protect from adverse nasal and pulmonary responses to airway allergen provocation. METHODS: Ovalbumin (OVA)-sensitized Brown Norway rats were treated orally with gammaT before intranasal provocation with OVA. Twenty-four hours after two challenges, histopathological changes in the nose, sinus and pulmonary airways were compared with gene expression and cytokine production in bronchoalveolar lavage fluid and plasma. RESULTS: We found that acute dosing for 4 days with gammaT was sufficient to provide broad protection from inflammatory cell recruitment and epithelial cell alterations induced by allergen challenge. Eosinophil infiltration into airspaces and tissues of the lung, nose, sinus and nasolacrimal duct was blocked in allergic rats treated with gammaT. Pulmonary production of soluble mediators PGE(2), LTB(4) and cysteinyl leukotrienes, and nasal expression of IL-4, -5, -13 and IFN-gamma were also inhibited by gammaT. Mucous cell metaplasia, the increase in the number of goblet cells and amounts of intraepithelial mucus storage, was induced by allergen in both pulmonary and nasal airways and decreased by treatment with gammaT. CONCLUSIONS: Acute treatment with gammaT inhibits important inflammatory pathways that underlie the pathogenesis of both AR and asthma. Supplementation with gammaT may be a novel complementary therapy for allergic airways disease.

Acetyl-L-carnitine and alpha-lipoic acid supplementation of aged beagle dogs improves learning in two landmark discrimination tests.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.

Bacterial defenses against oxidative stress.

Bacteria treated with low doses of oxidants such as hydrogen peroxide adapt to subsequent high doses of these oxidants by inducing the expression of numerous genes. The study of these genes and the roles they play in defending bacteria against oxidative damage has given general insights into what oxidants are hazardous to cells, what cell constituents are damaged by oxidants, and how cells sense and respond to oxidative stress.

Detection of mutagenic impurities in carcinogens and noncarcinogens by high-pressure liquid chromatography and the Salmonella/microsome test.

We have used high-pressure liquid chromatography and the Salmonella/microsome mutagenicity test to look for mutagenic impurities in 11 carcinogens and noncarcinogens. Because of the million-fold range in mutagenic potency observed in the Salmonella test, even trace amounts of potent mutagenic impurities in a nonmutagenic compound could be detected. The mutagenicity of 7-hydroxy-2-acetylaminofluorene, a noncarcinogen in the standard animal carcinogenicity tests, is shown to be due to a small amount of impurity, which is probably the potent carcinogen 2-acetylaminofluorene. This is discussed in relation to the statistical limitations of animal carcinogenicity tests. We also discuss the role of mitogenic impurities in assessing the mutagenicity of environmental (and industrial) chemicals with high-sensitivity mutagenicity assays, such as the Salmonella/microsome test.

Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test.

Seventeen cancer chemotherapeutic agents were tested for their ability to mutate Salmonella typhimurium tester strains in the Salmonella/microsome mutagenicity test. There was a high correlation between the mutagenicity and carcinogenicity of a given agent. Carcinogens positive in the test were Adriamycin, daunomycin, 1-propanol-3,3'-iminodimethanesulfonate, cyclophosphamide, isophosphamide, hycanthone, chlornaphazin, nitrogen mustard, uracil mustard, melphalan, and thio-tepa. Two carcinogesn, actinomycin D and bleomycin, were not detected as mutagens. The presumptive noncarcinogen, methotrexate, was negative in the test. Tilorone and 6-mercaptopurine, tentatively classified as noncarcinogens, were mutagenic. The carcinogenicity of cis-dichlorodiammineplatinum(II), which was positive in the test, has not been determined.

Immunochemical identification of a tRNA-independent cytokinin-like compound in Salmonella typhimurium.

Chemical and immunological characterization of Salmonella typhimurium cell extracts indicates that this organism produces a molecule which closely resembles the plant growth regulator, cytokinin. Alcohol-soluble cationic ultraviolet-absorbing material was fractionated by reverse-phase HPLC using gradient conditions optimized previously for modified nucleoside separation. A single hydrophobic compound was identified in the cytokinin region of the gradient, and limited quantities of the compound were prepared by HPLC fractionation of crude extracts. The compound demonstrated significant activity in a radioimmunoassay for cytokinins which detects N6-isopentenylated adenine derivatives. Boronate affinity chromatography indicated the compound is likely to be ribosylated and therefore a nucleoside. These and other tests indicate the compound has the most notable structural characteristics of a cytokinin. Spectral analysis and chromatographic comparison with cytokinin standards indicate the compound also has some unique structural features. Presence of the compound in extracts of an S. typhimurium mutant blocked for synthesis of tRNA-derived cytokinins excluded tRNA as a source for the compound and implicates existence of a tRNA-independent pathway for cytokinin biosynthesis in this bacterial species.

Sequence specificity of tRNA-modifying enzymes. An analysis of 258 tRNA sequences.

The specificity and recognition of tRNA-modifying enzymes may be accounted for in part by nucleotide sequences which are localized next to the modifiable nucleoside. In order to determine the sequence specificity of tRNA-modifying enzymes, we have surveyed 55 published tRNA sequences from Escherichia coli, Salmonella typhimurium and T4 phage. For each modified nucleoside, the nucleotide sequence surrounding the modification site was determined for all tRNAs known to contain the modified nucleoside. Subsequently all tRNAs not containing the modified nucleoside were examined for the absence of the putative recognition site. We present the detailed analysis of 12 modified nucleosides for which we found a strong correlation between the modified nucleoside and the local nucleotide sequence. This suggests that these sequences may be recognition sites for tRNA-modifying enzymes. For each of the 12 modified nucleosides we have identified a recognition sequence present in the tRNA set containing the modification and not in the set without it. All 203 other published tRNA sequences were then examined to see if the sequence specificity rules apply to other organisms, including both prokaryotes and eukaryotes. In several cases a good adherence was found, indicating conservation of the putative recognition sequences.

Mitochondrial decay in aging.

Several mitochondrial functions decline with age. The contributing factors include, the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning.

Plasma and urinary levels of malondialdehyde-like products (MDA) and isoprostanes were identified as markers of in vivo lipid peroxidation in an animal model of CCl4 poisoning. We sought to determine the extent to which the formation of these oxidation products is influenced by inhibition of the cyclooxygenase enzymes which catalytically generate proinflammatory lipid peroxidation products known as prostaglandins and thromboxane. In the present studies, after induction of oxidant stress in rats with CCl4, lipid peroxidation products measured in plasma and urine demonstrate that isoprostanes and MDA can be partially inhibited by cyclooxygenase inhibitors, albeit to different extents. The lowering of isoprostane and MDA formation, however, may not to due primarily to the diminution of catalytic generation of isoprostanes or MDA by the cyclooxygenases but, rather, may be the result of the suppression of nonenzymatic lipid peroxidation. This is suggested since 8,12-iso-iPF2alpha-VI is also reduced by indomethacin, yet, unlike other isoprostanes and MDA, it is not generated catalytically by the cyclooxygenase. Thus, although the two cyclooxygenase inhibitors we tested have statistically significant effects on the measurements of both isoprostanes and MDA in this study, the results provide evidence that these lipid-degradation products primarily constitute markers of oxidative stress.