Delbert M. Shankel

Natural antimutagenic agents

Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the authors laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Multiple drug resistance

Multiple drug resistance to antibacterial agents, antifungals, antivirals, antiprotozoals, and antitumor agents has risen spectacularly in the last decade or so and presently threatens eventually to put an end to successful chemotherapy in all of the above fields. This review summarizes the known origins of the problem, its present dimensions, the means employed to combat the phenomenon and promising avenues for future developments.

Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (west Africa).

Organic solvent extracts of leaves of 4 common edible vegetable plants--Bryophyllum pinnatum, Dialium guincense, Ocimum gratissimum and Vernonia amygdalina--had inhibitory activity for His- to His+ reverse-mutations induced by ethyl methanesulfonate acting on Salmonella typhimurium TA100. The concentrated ethyl acetate, methanol and petroleum ether extracts were heat-stable when dissolved in dimethyl sulfoxide. The Bryophyllum ethyl acetate extract was fractionated into alkaloidal/water-soluble, acids, polar lipid and non-polar lipid fractions. The polar and non-polar lipid fractions inhibited reversion mutations induced by ethyl methanesulfonate acting on TA100 or TA102, and were also active against reversions induced by 4-nitro-O-phenylenediamine and 2-aminofluorene in TA98. The alkaloidal/water-soluble and the acid fractions had no appreciable antimutagenic activities.

Isolation and Identification of Higher Plant Agents Active in Antimutagenic Assay Systems: Glycyrrhiza Glabra

The reproduction and maintenance of identity of species are biological imperatives, and it is not surprising that there exist mechanisms to minimize or repair the deleterious influence of noxious chemicals in the environment on DNA. Animals tend to defend themselves through the use of enzymes which intercept aggressive chemicals and convert them to less dangerous substances. Animal cells also contain a variety of preformed smaller molecular weight chemicals which can react with oxidized species and free radicals and convert them to less virulent electrophiles (1). It is now becoming clear that higher plants also contain a variety of preformed secondary metabolites which represent a structurally diverse array of antimutagenic and desmutagenic compounds (6). Many, but not all, would appear to be enzyme inhibitors or antioxidants. Since a number of plant constitu-ents are mutagenic (18), it seems reasonable that higher plants should also contain molecules capable of antimutagenicity so as to survive the effects of their own metabolism. Study of such substances has the potential of revealing much interesting molecular detail about the processes of mutagenesis and antimutagenesis. A rather more distant hope is that such substances might be safe enough to provide protection for individuals perceived to be at risk. This would appear to be the case with a number of minor anticarcinogenic constituents consumed as part of our diet (4, 20).

Antimutagenicity of secondary metabolites from higher plants

Higher plants contain both mutagens and antimutagens and are susceptible to mutagenesis but screening programs for detection of antimutagenesis rarely employ higher plant systems. Short-term bacterial and mammalian tissue culture systems are the norm. Using modified screening tests for detecting antimutagenic agents, higher plants have been shown to contain a variety of structurally novel antimutagenic agents. Systematic bioassay-directed methodology resulted in the isolation in pure form and biological and chemical characterization of the responsible individual active components from various plants. The methodology in use is illustrated by the isolation of cinnamic acid, cinnamyl cinnamate and cinnamyl ricinoleate as the active constituents of the classic medicinal plant product, Styrax asiatica. The methods which may be used to reveal structure-activity relationships and to explore putative molecular modes of action are illustrated with excerpts from the same study.

Inhibition of dark repair of ultraviolet damage in DNA by caffeine and 8-chlorocaffeine. Kinetics of inhibition

SummaryThe inhibition of dark repair by caffeine and 8-chlorocaffeine was investigated by a cellular enzyme kinetics system. The results suggest that the caffeine and 8-chlorocaffeine act by inhibiting the excision enzyme of the dark repair system.

The ability of certain antimutagenic agents to prevent development of antibiotic resistance.

Resistance to multiple antimicrobial agents has now become a prominent fact of contemporary life. It is believed that poor patient compliance, e.g. interrupted or premature cessation of therapy; and misuse or abuse of antibiotics, e.g. wrong antibiotic or insufficient dose, play important roles in resistance development. We present evidence that, this form of resistance often stems from spontaneous mutations accompanied by the positive selecting pressure of the doses of antibiotics being between the MIC and MBC levels. A number of antimutagenic agents, e.g. green tea catechins, and other antioxidants, etc. are able to suppress the emergence of resistance. In many cases, these agents are capable of exerting these effects at doses which by themselves produce no visible effect on growth. In a number of cases antimutagenic substances capable of preventing resistance emergence are present in normal food stuffs. These effects are exerted against resistance to tetracyclines, fluoroquinolones, macrolides, beta-lactams, aminoglycosides and the like. The implications of these laboratory findings for practical chemotherapy are discussed.

Antimutagenic activity of extracts from Japanese eggplant

Using the Salmonella/microsome assay, the antimutagenic effects of specific components of the extracts from eggplant fruits were investigated. The eggplant fruit juice exhibited an antimutagenic activity against 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) induced mutagenicity. In some of the fractions extracted with several organic solvents (acetone, petroleum ether, ethyl acetate; and methanol), the activity was recognized. No mutagenicity or toxicity for Salmonella typhimurium TA98 in the presence of S9 mixture was observed with any of the extracts. It is suggested that there are multiple components of the activities that exist in the eggplant fruit. We isolated lutein from the 84% methanol (methanol/water, v/v) layer, pheophorbide or chlorophyllide from the 70% methanol layer and tannins containing sugar-moieties from the water layer. Pheophytin a and b, Mg-free derivatives of chlorophyll a and b, were isolated from the petroleum ether layer as possible antimutagens. The pheophytin a with S9 mix inhibited by 30-40% the mutagenicity of Trp-P-2.

The influence of analogs of xanthine on the mutagenic effect of ultraviolet light.

SummaryEight compounds can be added to those purine analogs which give synergistic effects with ultraviolet light: 1,3,7,9-tetra-methylxanthine methyl sulfate, 8-chlorocaffeine, ethyltheophylline, 6-dimethylaminopurine, 6-methoxypurine, 8-ethoxycaffeine, 1,3-dimethyl uric acid and 3-methyl uric acid. It appears likely that these are merely representatives of the class of methyl purines, and that other compounds are also active. The synergistic activity varies among the compounds investigated; some give a synergistic effect slightly higher than caffeine, but apparently act by the same mechanism. The height of effect of all of the synergistic purine analogs has been correlated with the stability of the structures determined by inductive effects and steric hindrance. The lack of activity with pyrimidines (l-methyl thymine and 1,3-dimethyluracil) leads to the conclusion that both rings (7,8,9 positions) of the purine molecule are necessary for the synergistic effect.

Effects of ethidium, quinacrine and hycanthone on survival and mutagenesis of UV-irradiated Hcr+ and Hcr- strains of E. Coli B/r.

Summary Ethidium, quinacrine or hycanthone when incorporated in the post-irradiation plating medium have all been found to decrease survival and increase str r mutation frequencies in a UV-irradiated E. coli B/r strain. The enhancement of mutational yield was shown, for quinacrine, to apply also to UV-induced trp - → Trp + reversions. Since these three compounds do not enhance mutational yields, and have at best only a weak antimutagenic action, in an Hcr - strain, it seems probable that ethidium, quinacrine and hycanthone are primarily inhibitors of excision repair.