Plant tannins inhibit the induction of aberrant crypt foci and colonic tumors by 1,2-dimethylhydrazine in mice.

We have shown that naturally occurring tannins possess antitumor promotion activity in mouse skin. In the present investigation, we studied the ability of a hydrolyzable tannin, gallotannin (GT), and a condensed tannin extracted from red alder (RA) bark to inhibit 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF) and tumors in Balb/c mice. In addition, we determined the ability of GT to inhibit the proliferation and to induce apoptosis in a human colon cancer cell line (T-84). Mice were given tannins by intraperitoneal injections, by gavage, or in drinking water before treatment with DMH for 24 weeks. Alternatively, mice were given tannins by intraperitoneal injection or gavage for only 2 weeks before DMH administration, then tannin administration was discontinued and mice were treated with DMH for 24 weeks. The multiplicity, size, and distribution of ACF and tumors were significantly inhibited by GT and RA in the above treatment regimens. The most effective treatments included GT by gavage, RA bark extract by intraperitoneal injection, and either tannin dissolved in drinking water. Extent of inhibition of ACF and tumors was gender independent. In cell culture experiments, GT treatment for three days inhibited the growth of T-84 cells, with a concentration resulting in half-maximal inhibition estimated to be 20 micrograms/ml. The treatment was not cytotoxic to cells at 1-40 micrograms/ml. Interestingly, at 10 micrograms/ml, GT induced apoptosis in T-84 cells as determined by the Hoechst DNA staining technique. Collectively, these findings support a potential role for tannins as chemopreventive agents against colon cancer.

Seasonal changes in the composition of the essential oil extract of East Mediterranean sage (Salvia libanotica) and its toxicity in mice.

Sage (Salvia libanotica) is an East Mediterranean plant, the extract of which is used for the treatment of colds, coughs, and stomach ache. Experimental studies on the toxicity of its oil are scarce despite its wide use in traditional medicine. This study aims to provide data on its acute toxicity and to investigate the relationship between seasonal changes in oil composition and toxicity. The composition of the oil extract from the leaves of this plant was determined at four different times of the year; August (summer), October (fall), January (winter) and April (spring). The toxicity of each fraction was investigated following intraperitoneal (i.p.) injection into mice. Distillations of oils from plants and GC analyses revealed that the main constituent of sage oil is 1,8-cineole. Other components included ketones such as camphor and alpha,beta-thujone, terpenes such as limonene and alpha,beta-pinene, and alcohols such as borneol and linalool. Major seasonal changes were found in the composition of the oil. Essential oil extracted from plants collected in the winter season (January) contained higher levels of camphor (12.3%), alpha,beta-thujone (1.9%), and camphene (4.8%). The winter extract was found to be the most toxic, (LD(50): 839 mg/kg body weight) and exhibited powerful convulsant properties. This indicates a strong correlation between the contents of camphor, thujones and camphene and the oils' toxicity. The spring extract was the least toxic (LD(50): 1200 mg/kg body weight) and contained lower levels of camphor (7.7%), alpha,beta-thujone (1.3%) and camphene (3.1%). Thus, we recommend that oil extracts of sage marketed for use in certain unconventional medicines be prepared from spring plants.

Quinoxaline 1,4-dioxides as anticancer and hypoxia-selective drugs.

Hypoxic cells which are found in solid tumors are resistant to anticancer drugs and radiation therapy. Thus, for effective anticancer chemotherapy, it is important to identify drugs with selective toxicity towards hypoxic cells. Quinoxaline 1,4-dioxides (QdNOs) are heterocyclic aromatic N-oxides that have been found to possess potent antibacterial activities (inhibit microbial DNA synthesis) especially under anaerobic conditions; thus they are under evaluation as bioreductive drugs for the treatment of solid tumors (1). We investigated the ability of four differently substituted QdNOs to inhibit cell growth and induce cell cycle changes in two human tumorigenic epithelial cell lines under oxic conditions. We also evaluated the toxicity of these drugs to cancer cells cultured under hypoxic conditions. Two epithelial cell lines (the T-84 human colon cancer-derived cell line, and the SP-1 keratinocyte cell line) were treated with various doses of the QdNOs and harvested at different times after treatment. Proliferation and cell cycle results showed a structure-function relationship in the activity of the various QdNO compounds with the 2-benzoyl-3-phenyl-6,7-dichloro-derivative of QdNO (DCBPQ) being the most potent cytotoxin and hypoxia-selective drug. The 2-benzoyl-3-phenyl (BPQ) and the 2-acyl-3-methyl-derivative of QdNO (AMQ) were less cytotoxic but arrested almost 50% of the cells in the G2M phase of the cell cycle at doses of 30 and 120 microM, respectively. The tetramethylene derivative of QdNO (TMQ) did not affect the growth and cycling of cells cultured in air and was the least potent cytotoxin to hypoxic cells. Our results indicate that the QdNOs are hypoxia-cytotoxic drugs whose activity varies according to the substituents on the quinoxaline 1,4-dioxide heterocycle. Because of their selective toxicity to hypoxic cells (cells found in human tumors), these drugs may provide useful therapeutic agents against solid tumors.

Tannins protect against skin tumor promotion induced by ultraviolet-B radiation in hairless mice.

We recently showed that Tarapod tannic acid (TA), a hydrolyzable tannin extracted from the pods of the Tara tree (Caesalpinia spinosa), was more effective than other tannins tested at inhibiting ultraviolet-B (UV-B)-stimulated hydrogen peroxide activity (an indirect measure of free radicals) in the skin of hairless mice. We also found that Tarapod TA inhibited UV-B-induced ornithine decarboxylase activity and UV-B-stimulated DNA synthesis, two biochemical markers linked to the skin tumor-promoting ability of this physical carcinogen. For this reason, we examined the effect of topical application, force feeding (gavage), and intraperitoneal injections of Tarapod TA on mouse skin chronically treated with UV-B light. Mice were initiated by a single topical application of 7,12-dimethylbenz[a]anthracene (50 nmol) and promoted by two weekly treatments with UV-B light (250 mJ/cm2) for 25 weeks. Topical application of Tarapod TA, 20 minutes before irradiation, resulted in a dose-dependent inhibition of tumor incidence (number of mice with tumors) and tumor yield (number of tumors/mouse), with 8 mg of TA inhibiting tumor yield by 70% at Week 25. Intraperitoneal injections of low doses (10 mg/kg mouse body wt), but not of high doses (25 mg/kg body wt), of TA afforded protection against UV-B-induced papillomas. However, the protection by intraperitoneal injection was lower than that observed by topical application: 10 mg/kg body wt of TA reduced tumor yield by 55%. The force feeding of 10 mg of Tarapod TA before irradiation failed to significantly inhibit the yield of tumors at the end of the experiment but delayed tumor appearance by six weeks. These results suggest that plant tannins administered topically or injected intraperitoneally reduce the tumor-promoting effects of UV-B radiation and thus could be useful photoprotectants.

Chemopreventive effects of sage oil on skin papillomas in mice.

Salvia libanotica (sage) extract is a popular plant remedy used by Middle Eastern people to treat common complaints such as colds and abdominal pain. In this study, the chemopreventive effects of sage oil on 7,12 dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin papillomas was investigated. Furthermore, its growth inhibitory and cytotoxic effects on a mouse papilloma-derived cell line (SP-1) were studied using 3H-thymidine incorporation, cell count and trypan blue dye exclusion assays. Sage oil was either applied topically to mouse skin at concentrations of 5, 50 and 100% in acetone, injected intraperitoneally at concentrations of 4 (37 mg/ml) and 8% (75 mg/ml) in saline or given by gavage at 100% twice per week for 20 weeks, 20 minutes prior to each promotion treatment with TPA. The topically applied 100% oil extract delayed tumor appearance by 4 weeks and inhibited tumor incidence and yield by 19 and 61%, respectively, at week 20. Topical application of 50% and 5% sage oil inhibited tumor yield by 41% at week 20. Tumor weight was decreased by 75% and 80% following treatment of mouse skin with 50% and 100% oil, respectively. Intraperitoneal injections and gavage treatments failed to inhibit the promotion of tumors in mouse skin, but significantly decreased tumor weight and volume. Sage oil displayed strong growth inhibitory effects on the SP-1 papilloma derived cell line following 24 hrs of treatment with estimated IC50 of 50 microg/ml. This observed growth inhibition was due to cytostatic and not cytotoxic effects. Our results suggest that the oil extract of the sage plant has potent suppressive activities against tumor promotion in mouse skin and thus could be an effective chemopreventive agent against skin cancer.

Plant tannins as inhibitors of hydroperoxide production and tumor promotion induced by ultraviolet B radiation in mouse skin in vivo.

The anti-oxidant and anti-tumor promotion activities of several tannins extracted from plants were examined in mouse skin treated with ultraviolet B (UVB) radiation in vivo. Hydroperoxide production was found to be maximally stimulated at a UVB dose of 200 mj/cm2, beyond which no further stimulation occurred. Treatment of mouse skin with two UVB doses of 225 mj/cm2 each, applied at a 48 h interval gradually increases the hydroperoxide (HPx)-producing activity of the epidermis, which is maximally stimulated at 4 days and returns to control levels at 15 days. The magnitude of the HPx response is found to increase with repeated UVB treatments applied at a 48 h interval and reaches a maximum level following four treatments. Of the three tannins tested (Commercial TA, Tarapod TA, and Oak TA), Tarapod TA is found to be the most effective inhibitor of UVB-stimulated HPx activity. Pretreatment with Tarapod TA inhibits, in a dose-dependent manner, this HPx response to UVB radiation. Inhibition by Tarapod TA occurs when it is applied at distant times before (-12 h) or after (+24 h) UVB radiation. When applied 20 min before UVB radiation, twice a week for 25 weeks, 8 mg of Tarapod TA inhibits the incidence and yield of papillomas promoted by UVB light in initiated skin by 34 and 70% respectively. Furthermore, when 10 mg/kg of mouse body weight of Tarapod TA was injected intraperitoneally, for a period of 25 weeks, 20 min prior to UVB treatment, it inhibited the yield of papillomas by 44%, suggesting that plant tannins when administered by various means are useful photoprotectants.

Inhibitory effects of plant tannins on ultraviolet light-induced epidermal DNA synthesis in hairless mice.

Naturally occurring hydrolyzable (HT) and condensed (CT) tannins and their monomeric units were tested for their ability to inhibit the stimulation of DNA synthesis by UVB radiation. Hairless mice were irradiated with either single (200 mJ/cm2) or multiple (150 mJ/cm2) doses of UVB applied at 24 h intervals and epidermal DNA synthesis was measured at different times after the last of these treatments. The peak of DNA synthesis that is observed 48-56 h after a single UVB irradiation shifts to an earlier time of 16-24 h after multiple UVB treatments. Interestingly, the early inhibitory period of DNA synthesis observed 8 h after a single UVB treatment is not detected following multiple UVB treatments. Rather, DNA synthesis is stimulated six-fold 24 h after multiple UVB treatment, a response that is higher than the peak occurring 48-56 h after a single UVB irradiation. The disappearance of the early period of inhibition when the peak of DNA synthesis shifts to an earlier time may be linked to reactive oxygen species brought to the epidermis by infiltrating leukocytes, which, in turn, act as second messengers to stimulate growth signals in cells. Topical applications of HT or CT remarkably inhibit the DNA responses to single and multiple UVB treatments, an effect that is dependent on the dose and time of administration. Indeed, the peak stimulation of DNA synthesis is maximally inhibited when 17 mg of Tarapod tannic acid (TA), an HT, are applied topically 20 min before a single UVB treatment. The polymeric tannins inhibited DNA synthesis to a greater degree than equal doses of their monomeric units, gallic acid and catechin. These results suggest that various oligomeric HT and CT may be useful against tumor-promoting responses associated with the exposure of skin to physical carcinogens.

Photoprotective effects of some quinoxaline 1,4-dioxides in hairless mice.

2-benzoyl-3-phenylquinoxaline 1,4-dioxide (BPQ) and other substituted quinoxaline 1,4-dioxides (QdO) were tested for their ability to inhibit the stimulations of ornithine decarboxylase (ODC) enzyme activity and DNA synthesis, two biochemical markers linked to skin tumour promotion by ultraviolet B (UVB) radiation. Topical application of BPQ on the dorsal skin of hairless mice was found to inhibit in a dose-dependent manner UVB-induced ODC activity and DNA synthesis. When applied 20 min before UVB radiation, a dose of 17 mg BPQ applied in 0.4 ml of vehicle inhibited UVB-induced ODC activity and DNA synthesis by 95% and 85%, respectively. This inhibitory effect is dependent on the time of administration of BPQ relative to UVB radiation, with a generally greater inhibition observed when this compound is applied before rather than after UVB treatment. The inhibitory abilities of the other QdO on the ODC and DNA responses induced by UVB radiation greatly varied and appear to be dependent on the structure of the compounds and their metabolic activation in the skin following irradiation. The remarkable effectiveness of BPQ against the ODC and DNA markers of UVB promotion is also observed following multiple applications of this agent. These results suggest that QdO, in particular BPQ and certain derivatives of it, may be useful in protecting the skin against UVB-induced skin damage.

Inhibition of ultraviolet-B radiation induced ornithine decarboxylase activity and edema formation by hydrolyzable and condensed tannins in mouse skin in vivo.

Naturally occurring hydrolyzable (HT) and condensed (CT) tannins and their monomeric units were tested for their ability to inhibit the induction of epidermal ODC activity and the formation of skin edema by UVB, two responses that are linked to the hyperplastic and inflammatory components of skin tumor promotion by this agent. Hairless mice were irradiated with either single (200 mJ/cm2/sec) or multiple (150 mJ/cm2/sec) doses of UVB and epidermal ODC activity was assayed at different times following irradiation. The peak of ODC induction which is observed 30-40 hours after a single UVB irradiation increases by 2.5 fold and shifts to a much earlier time of 5 hours after two UVB treatments repeated at 24-hour intervals. Topical applications of the various plant tannins, before or after irradiation, were found to inhibit, in a dose-dependent manner, epidermal ODC activity induced by single and multiple UVB treatments. Furthermore, the various HT and CT samples resulted in significant protection against UVB radiation-caused cutaneous edema. In general, the polymeric tannins inhibited ODC induction and edema to a greater degree than equal doses of their monomeric units, gallic acid and catechin. These results, in conjunction with our prior publications, suggest that various HTs and CTs may be useful against the hyperplastic and inflammatory responses associated with the exposure of skin to the tumor-promoting effects of both physical and chemical environmental carcinogens.