Hans F. Stich

The beneficial and hazardous effects of simple phenolic compounds

The current emphasis on screening the environment for man-made genotoxic and carcinogenic compounds detracts from studies on the possible health hazard or beneficial effects of naturally occurring agents to which humans are exposed daily. The simple phenolics, which are ubiquitous among plants, used as food additives, and ingested daily in milligram quantities, belong to this category of compounds. They induce double-strand DNA breaks. DNA adducts, mutations and chromosome aberrations in a great variety of test systems. However, they can suppress the genotoxic activity of numerous carcinogenic compounds in both in vitro and in vivo assays. This dual function of dietary phenolics also becomes evident when their carcinogenic or anticarcinogenic potential is examined. Some, but not all, phenolics induce precancerous lesions, papillomas and cancers, act as cocarcinogens, and exert a promoting effect in various rodent assays. On the other hand, phenolics have proved to be potent inhibitors of carcinogenesis at the initiation and promotion stages induced by carcinogens and promoters of different molecular structures. The extent to which a health hazard or protective activity of complex dietary mixtures is due to their phenolic content remains an unresolved issue. In addition, these multiple, occasionally contradictory functions of simple phenolics make it difficult to propose their use as chemopreventive agents.

Response of oral leukoplakias to the administration of vitamin A

Tobacco/betel nut chewers (Kerala, India) with well-developed oral leukoplakias were chosen for a short-term intervention trial of vitamin A therapy. Participants were randomly distributed into two groups, one receiving 200,000 IU vitamin A per week (0.14 mg/kg body wt/per day) for 6 months, and the other receiving placebo capsules. Their cancer-causing habit, which can be quantitated (an average of 13.1 betel quids/day, 26.1 min/quid), did not change during the trial period. The 6-month oral administration of vitamin A caused complete remission in 57.1% of participants, and a total suppression of the development of new leukoplakias in all chewers receiving vitamin A (n = 21), as compared to 3% and 21%, respectively, in the placebo group (n = 33). The results were substantiated by examining the histological and cytological changes on small biopsies which were taken at the onset and at the completion of the trial period. Over the 6-month period of vitamin A administration, the number of layers of spinous cells decreased in 85% of the participants, the loss of polarity of basal cells was reduced from 72.2% to 22.2% of chewers, subepidermal lymphocytic infiltration was greatly diminished from 66.7% to 5.5% of chewers, and nuclei with condensed chromatin disappeared from the epidermal layer (72.2% before to 0% at the end of the trial).

DNA repair and chromatid anomalies in mammalian cells exposed to 4-nitroquinoline 1-oxide

Abstract The oncogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO) induces DNA-repair synthesis (unscheduled DNA synthesis) in diploid, aneuploid, normal and neoplastic human and Syrian-hamster cells. DNA-repair synthesis occurs in nuclei at G1, G2 and S-phase and in metaphase chromosomes of Syrian-hamster cells exposed to 4NQO. DNA-repair synthesis was separated from DNA-replication synthesis associated with chromosome replication at S-phase by arginine deprivation. The degree of [ 3 H]TdR incorporation into nuclear DNA is dependent on the dose of 4NQO (5·10 −8 to 1·10 −5 M ) and on the amount of DNA per cell. The time course of DNA-repair synthesis induced by 4NQO or UV was examined on non-dividing cells which were arrested by an arginine-deficient culture medium: an early occurring peak is followed by an abrupt decline at about 8 h post-treatment which is succeeded by a prolonged low level incorporation of [ 3 H]TdR. The effect of a completed and uncompleted repair synthesis on the flow of cells into S-phase and on the frequency of chromosome anomalies was studied on cells arrested by arginine deprivation and triggered to divide by addition of arginine. There appears to be a relationship between the degree of repair synthesis and on the other hand frequency of cells entering S-phase, incidence of metaphase plates with chromatid breaks, flow of cells from G2 into pro-metaphase and “uncoiling” of metaphase chromosomes or heterochromatic segments of interphase nuclei.

Naturally Occurring Phenolics as Antimutagenic and Anticarcinogenic Agents

Epidemiological evidence points to an inverse relationship between the consumption of vegetables and the incidence of cancer at various sites (Hirayama, 1979, 1981; Graham et al., 1978; Mettlin et al., 1981). The search for the protective components in these vegetables has focused on B-carotene and vitamin A (e.g., Bjelke, 1975; Shekelle et al., 1981; Cambien et al., 1980; Peto et al., 1981; Doll and Peto, 1981; Marshall et al., 1982) and ascorbic acid (e.g., Haenszel and Correa, 1975; Kolonel et al., 1981). However, the inverse relationship observed between the ingestion of green/yellow vegetables and the incidence of human cancers could conceivably be due to many other plant components. At present, the percentage contribution of vitamins to the cancer-protective activity of vegetables or fruits is unknown. In this paper, we present results suggesting an involvement of naturally occurring phenolics in the prevention of genotoxicity and carcinogenicity. Since the number of phenolics in various plants is staggering and the discussion of their beneficial or toxic effects is beyond the scope of any short review, we have focused on non-flavonoid simple phenolics (C6), phenolic acids (C6-C1), cinnamic acid and related compounds (C6-C3).

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

Abstract The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens. The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N -oxides was compared to that induced by the mutagen and carcinogen N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) and UV-irradiation. The XP cells showed ( 1 ) a reduced level of DNA repair synthesis when exposed to various carcinogenic N -oxides, ( 2 ) no unscheduled DNA synthesis following 6NQO and ( 3 ) a normal degree of DNA repair synthesis after treatment with MNNG. When the clone-forming capacity was examined the XP cells exhibited ( 1 ) a higher increased sensitivity to the various carcinogenic N -oxides, ( 2 ) no reduction in the clone formation following 6NQO and ( 3 ) a sensitivity virtually comparable to that of normal cells after treatment with MNNG. The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.

The involvement of reactive oxygen species in oral cancers of betel quid/tobacco chewers

Most biological reactions, including carcinogenesis, are complex processes involving thousands of compounds, their metabolites and intermediates. The separation of events which form part of a direct chain leading to neoplastic transformation from those which are mere by-products is a herculean task. In this study, we focused on the pros and cons of reactive oxygen species (ROS) being involved in the development of oral cancer among chewers of tobacco and areca nuts. The results revealed that bursts of ROS generation occur at different stages of carcinogenesis, and are caused by different mechanisms. This observation may have considerable practical implications. Different strategies will be required in the administration of chemopreventive agents in order to trap ROS formed in the alkaline (due to the addition of slaked lime) chewing mixture within the saliva of a chewer, to scavenge ROS within mucosal cells exposed to an array of tobacco- or areca nut-related carcinogens or tumour promoters, and to inhibit the action of ROS released from ROS-generating white cells during lymphocytic infiltration of the oral mucosa at a precancerous stage. The remission of oral leukoplakias following the administration of vitamin A (200,000 IU/week) or vitamin A (100,000 IU/week) plus beta-carotene (180 mg/week) for 6 months, the inhibition of new leukoplakias during this trial period, and the reduction of micronucleated oral mucosal cells in chewers treated with beta-carotene or vitamin A are indeed promising results. However, a better understanding of the role of ROS in various stages of carcinogenesis will provide the basis for selection of the proper chemopreventive agents and the design of a treatment regime which may either prevent the formation of precancerous lesions, induce their remission, or inhibit the progression of precancerous lesions into malignant cancers.

The action of transition metals on the genotoxicity of simple phenols, phenolic acids and cinnamic acids

Simple phenols (catechol, 4-methyl catechol, resorcinol, phloroglucinol and pyrogallol), phenolic acids (p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, gallic acid, syringic acid and salicylic acid), a phenylacetic acid (3,4-dihydroxyphenylacetic acid) and eugenol were assayed for clastogenic activity in Chinese hamster ovary (CHO) cells with and without the addition of a n S9 mixture, Cu2+ (10-4M) and Mn2+ (10-4M). All dihydroxylated and trihydroxylated phenolics induced chromatid breaks and exchanges. The introduction of a methyl group seems to reduce the clastogenic capacity. The addition of an S9 mixture or the transition metals Cu2+ and Mn2+ enhanced the chromosome-damaging activity in some phenolics and suppressed it in others.

ADAPTATION OF THE DNA‐REPAIR AND MICRONUCLEUS TESTS TO HUMAN CELL SUSPENSIONS AND EXFOLIATED CELLS*

In vitro assays for genotoxicity have been applied to over 11,000 chemicals, many of which have been found to have the capacity to induce mutations, chromosome aberrations, sister chromatid exchanges, mitotic recombination, gene conversion, nondisjunction, DNA fragmentation, DNA repair, and a host of other nuclear anomalies. The results of these observations have profoundly changed our attitude toward environmental carcinogenesis. Only two decades ago, chemicals with carcinogenic and mutagenic properties were considered to be rare. The induction of a DNA alteration and mutation in somatic cells was thought to be an extremely rare event. Today, one has the impression that we are living in a brew of carcinogenic and genotoxic compounds and that hundreds, if not thousands, of mutagens and clastogens enter man daily through the regular diet. Previously, the development of cancer was thought to occur in individuals accidentally exposed to an excessive dose of one carcinogen. Now one is inclined to accept the idea that, since relatively large amounts of carcinogens enter man and carcinogen-DNA adducts are formed in many organs, cancer will develop primarily in individuals who ingest too small an amount of anticarcinogenic agents or who have a defective defense mechanism. If this assumption is correct, then in vitro tests, which lack the complete metabolic activation and inactivation systems of a whole organism, may predict a potential but not the actual carcinogenic hazard to man. The design, development, and validation of short-term in vivo tests that are applicable to man are needed to obtain a higher and more reliable predictive value.

Clastogenic activity of caffeic acid and its relationship to hydrogen peroxide generated during autooxidation.

Caffeic acid is a clastogenic cinnamic acid found in a conjugated form in a variety of foods. The possibility that the biological activity of caffeic acid is due to hydrogen peroxide generated during its autooxidation in solution was investigated using chromosome aberrations in Chinese hamster ovary cells as a test system. Freshly prepared caffeic acid at pH 7.00 contained only traces of hydrogen peroxide, as assayed by the molybdate-catalyzed release of I-3. Such solutions exhibited clastogenic activity which could not be accounted for by the level of hydrogen peroxide present, and which was not significantly diminished by the addition of catalase or horseradish peroxidase. 3-day-old solutions of caffeic acid exhibited higher levels of hydrogen peroxide, and increased biological activity. In such solutions, the clastogenic activity was catalase-sensitive and could be entirely accounted for by the level of hydrogen peroxide present.

A comparative genotoxicity study of chlorogenic acid (3-O-caffeoylquinic acid)

Chlorogenic acid, a compound which occurs naturally in many food items, was assayed for genotoxic activity in 3 different test systems: reverse mutations in the preincubation test with Salmonella typhimurium, gene conversion with Saccharomyces cerevisiae strain D7, and chromosome aberrations in Chinese hamster ovary (CHO) cells. Chlorogenic acid was directly convertogenic and clastogenic, but lacked a mutagenic capacity in the Salmonella bioassay. The transition metal Mn2+ enhanced the clastogenic and convertogenic activity of chlorogenic acid. In the presence of Mn2+ (10(-4)M), chlorogenic acid increased the frequency of his+ revertants in TA98 and TA100 strains of S. typhimurium. Caffeic acid and, to a lesser degree, quinic acid, which are components of chlorogenic acid, also showed genotoxic activity. The results show the importance of using several assays in combination with transition metals when testing for genotoxicity.