Efficacy of crocin and safranal as protective agents against genotoxic stress induced by gamma radiation, urethane and procarbazine in mice.

Crocin (CRO) and safranal (SAF) are bioactive constituents of saffron (dried stigma of Crocus sativus flower), an expensive spice with medicinal properties. Aqueous extract of saffron is known for its antigenotoxic effect against environmental genotoxins/carcinogens. However, there is need to identify saffron constituents responsible for this antigenotoxic effect. The aim of our investigation was to ascertain the role of CRO and SAF as inhibitors of in vivo genotoxic stress. For this purpose, Swiss albino mice were pretreated with CRO (50-mg/kg body weight (bw))/SAF (0.025- and 0.25-ml/kg bw) by gavage for 2 days. Thereafter, the pretreated mice were exposed to the genotoxic agents: (1) gamma radiation (GR; 2 Gy), (2) urethane (URE; 800 mg/kg) and (3) procarbazine (PCB; 60 mg/kg). In addition, CRO (50 mg/kg) was co-administered with the nitrosation reaction mixture of methylurea (MU; 300-mg/kg bw) + sodium nitrite (15 mg/kg) which can form N-nitroso-N-MU in the stomach. Genotoxic damage was measured by performing the bone marrow micronucleus test. Results obtained demonstrated significant reductions in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of mice pretreated with CRO/SAF before exposure to the above DNA damaging agents, GR, URE and PCB. Co-administration of CRO with the nitrosation reaction mixture led to significant decrease in genotoxicity when compared to nitrosation reaction mixture alone. Histopathological studies revealed that these saffron constituents reduced testicular cell damage induced by the test genotoxins. The cell-free DNA-nicking assay using pBR322 DNA showed significant protective effects of CRO against hydroxyl radical-induced strand breaks.

Ocimum sanctum Linn. (Holy Basil) ethanolic leaf extract protects against 7,12-dimethylbenz(a)anthracene-induced genotoxicity, oxidative stress, and imbalance in xenobiotic-metabolizing enzymes.

The present study was designed to evaluate the protective effects of ethanolic Ocimum sanctum leaf extract against 7,12-dimethylbenz[a]anthracene (DMBA)-induced genotoxicity, oxidative stress, and imbalance in xenobiotic-metabolizing enzymes. Four different concentrations of ethanolic O. sanctum leaf extract (100, 200, 300, and 400 mg/kg of body weight) were administered to Wistar rats by intragastric intubation for five consecutive days followed by intraperitoneal injection of DMBA (35 mg/kg of body weight) 90 minutes after the final dose of the extract. Administration of DMBA increased bone marrow micronuclei, phase I enzymes, lipid peroxidation, and protein carbonyl formation. This was accompanied by a significant decrease in the activities of phase II detoxification enzymes and antioxidants in the liver, erythrocytes, and bone marrow. Pretreatment with ethanolic O. sanctum leaf extract at a concentration of 300 mg/kg of body weight significantly reduced micronuclei formation and phase I enzymes as well as lipid and protein oxidation with enhanced antioxidant and phase II enzyme activities. The results of the present study suggest that ethanolic O. sanctum leaf extract inhibits DMBA-induced genotoxicity and oxidative stress by modulating xenobiotic-metabolizing enzymes, reducing the extent of lipid and protein oxidation and up-regulating antioxidant defenses.

Protective effect of black tea polyphenols against 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress during hamster buccal pouch carcinogenesis.

ABSTRACT This study was designed to evaluate the protective effect of black tea polyphenols (Polyphenon B) against genotoxicity and oxidative stress during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Since the bone marrow reflects hematopoietic stress caused by tumor cells, we used the frequency of micronuclei, the extent of lipid peroxidation, and the status of antioxidants in the bone marrow plasma as intermediate biomarkers of oxidative stress. All the hamsters painted with DMBA alone for 14 weeks developed buccal pouch carcinomas with a 75.4% increase in the incidence of bone marrow micronuclei as compared to untreated control (group 4). This was accompanied by an increase in lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) (61.3% and 17.8%, respectively) and a decrease in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) and the activities of GSH-dependent enzymes glutathione peroxidase (GPx) and glutathione S-transferase (GST) by 32.6%, 35.9%, and 62.8%, respectively, as compared to untreated control. Dietary administration of Polyphenon B significantly suppressed DMBA-induced HBP carcinomas by 20% and reduced the frequency of bone marrow micronuclei by 57.3% and TBARS and LOOH by 38.8% and 25.8%, respectively, compared to group 1 animals with significant elevations in the GSH:GSSG ratio (16.0%) and activities of GPx (29.8%) and GST (53.8%). Our results reveal that dietary supplementation of Polyphenon B exerts protection against DMBA-induced genotoxicity and oxidative stress by augmenting bone marrow antioxidant defense mechanisms.

Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agents in mice.

The genotoxic potential of anti-tumor drugs limits their efficacy in the treatment of cancers. Since ancient times, saffron (dried stigmas of Crocus sativus L.) has been used as a spice and medicinal herb. Saffron is a rich source of carotenoids and is known for its anti-cancer and anti-tumor properties. The present study was designed to ascertain the chemoprotective potential of saffron against the genotoxicity of three well-known anti-tumor drugs-cisplatin (CIS), cyclophosphamide (CPH) and mitomycin-C (MMC)--using comet assay. Three doses of saffron (20, 40 and 80 mg/kg b.w.) were orally administered to mice for five consecutive days prior to the administration of anti-tumor drugs under investigation. Pre-treatment with saffron significantly inhibited anti-tumor drugs induced cellular DNA damage (strand breaks) as revealed by decreased comet tail length, tail moment and percent DNA in the tail. These findings, together with our previous results, suggest a potential role for saffron as an anti-genotoxic, anti-oxidant and chemopreventive agent and could be used as an adjuvant in chemotherapeutic applications.

Comparative evaluation of the chemopreventive efficacy of green and black tea polyphenols in the hamster buccal pouch carcinogenesis model.

OBJECTIVES: To evaluate the comparative chemopreventive efficacy of green tea polyphenols (Polyphenon-E) and black tea polyphenols (Polyphenon-B) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. DESIGN AND METHODS: Hamsters were divided into 6 groups. Animals in group 1 served as controls. Animals in groups 2 and 3 were administered 0.05% Polyphenon-E and B, respectively, in the diet. The right buccal pouches of animals in groups 4-6 were painted with 0.5% DMBA three times a week for 14 weeks. While group 4 received no further treatment, hamsters in groups 5 and 6 received diet containing 0.05% Polyphenon-E and B, respectively. The status of carcinogen-metabolising enzymes, lipid peroxidation and glutathione-dependent antioxidants in the buccal pouch and liver, as well as the frequency of bone marrow micronuclei were used as biomarkers. RESULTS: Application of DMBA induced HBP carcinomas, increased genotoxicity with an imbalance in carcinogen-metabolising enzymes and the cellular redox status. Inhibition of HBP carcinomas by Polyphenon-E and B was associated with a significant decrease in phase I enzymes, modulation of lipid peroxidation and enhanced antioxidant and phase II enzyme activities. CONCLUSION: The greater efficacy of Polyphenon-B in inhibiting HBP carcinogenesis suggests that it may have a major impact in the chemoprevention of oral cancer.

Attenuation of N-methyl-N'-nitro-N-nitrosoguanidine induced genotoxicity and oxidative stress by tomato and garlic combination.

The protective effect of pretreatment with tomato and garlic against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced genotoxicity and oxidative stress was investigated in male Swiss mice. In vivo bone marrow micronucleus test was performed to assess the antigenotoxic effect of tomato and garlic. Oxidative stress was monitored by estimating the extent of lipid peroxidation and the status of the glutathione redox cycle antioxidants. Increased frequency of bone marrow micronuclei with enhanced lipid peroxidation was associated with compromised antioxidant defenses in MNNG treated animals. Although pretreatment with tomato and garlic significantly reduced the frequencies of MNNG-induced bone marrow micronuclei, the combination of tomato and garlic exerted a greater protective effect. This was associated with modulation of lipid peroxidation as well as reduced glutathione (GSH) and the GSH-dependent enzymes glutathione peroxidase (GPx) and glutathione-S-transferase (GST). These findings suggest that a diet containing even low levels of different naturally occurring compounds is effective in exerting antigenotoxic effects by modulating oxidative stress.

Tomato and garlic by gavage modulate 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice.

Chemoprotection by dietary agents is a promising strategy for cancer prevention. The aim of the present study was to evaluate the combined effect of tomato and garlic against 7,12-dimethylbenz-[a]anthracene (DMBA)-induced genetic damage and oxidative stress in 12-14-week-old male Swiss albino mice. The animals were randomized into experimental and control groups and divided into eight groups of five animals each. Group 1 animals were injected intraperitoneally with 35 mg/kg body weight DMBA suspended in peanut oil as a single dose. Groups 2-4 animals received tomato (500 mg/kg body weight), garlic (125 mg/kg body weight) and a combination of tomato and garlic for 5 days by gavage, respectively, followed by DMBA 1.5 h after the final feeding. The doses of tomato and garlic correspond to the average human daily consumption. Animals in groups 5, 6 and 7 received tomato alone, garlic alone and tomato + garlic combination, respectively, for 5 days. Group 8 animals received the same volume of water and served as control. The incidence of bone marrow micronuclei and the extent of lipid peroxidation and the concentrations of antioxidants glutathione, glutathione peroxidase and glutathione-S-transferase were measured in the liver, 48 h after DMBA exposure. Increased frequency of micronuclei and enhanced lipid peroxidation accompanied by compromised antioxidant defenses were observed in DMBA-treated animals. Although pretreatment with tomato or garlic significantly reduced the frequency of DMBA-induced bone marrow micronuclei, the combination of tomato and garlic exhibited more profound effect in inhibiting DMBA-induced genotoxicity and oxidative stress. We suggest that a broad spectrum of antimutagenic and anticlastogenic effects can be achieved through an effective combination of functional foods such as tomato and garlic.

Coffee-mediated protective effects against directly acting genotoxins and gamma-radiation in mouse lymphoma cells.

The cytokinesis-block micronucleus test was performed using L5178Y mouse lymphoma cells to ascertain whether or not standard (caffeinated) instant coffee, the commonly consumed polyphenolic beverage with antioxidant activity can protect against chromosomal damage induced by the directly acting agents N-methyl-N-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC), methyl methanesulfonate (MMS) and gamma radiation. Our results demonstrated significant reductions in the in vitro genotoxic effects of MNNG, MMC, and MMS following co-treatment of mouse lymphoma cells with standard instant coffee. Subsequently, the comet assay was carried out to assess the effect of coffee co-treatment on the level of DNA damage induced by MMS in mouse lymphoma cells. The results demonstrated a significant reduction in MMS-induced DNA damage following co-treatment with standard instant coffee. Protective effects were observed in mouse lymphoma cells which were treated with coffee immediately after exposure to gamma radiation (1 and 2 Gy). Another experiment showed protection when the mammalian cells were irradiated (0.5 and 1 Gy) midway (at 2 h) during a 4 h coffee treatment. However, the protective effect against the lower dose (0.5 Gy) was not significant. In addition we assessed the modulatory effect of coffee on MNNG-induced apoptotic frequency by flow cytometry. The results revealed only a minor influence of coffee on the frequency of apoptotic cells induced by the test compounds, rendering an increase in sensitivity for apoptosis as a reason for the reduced genomic damage an unlikely or at least incomplete explanation.

Anti-genotoxicity of coffee against N-methyl-N-nitro-N-nitrosoguanidine in mouse lymphoma cells.

The main aim of this work was to test the hypothesis that instant coffee, a commonly consumed polyphenolic beverage with antioxidant activity, can protect mammalian cells against genotoxic effects in vitro. For this purpose, the L5178Y mouse lymphoma cell line was selected to assess modulatory effects of coffee on the genotoxicity of N-methyl-N-nitro-N-nitrosoguanidine (MNNG). We initiated the work with a set of preliminary experiments in which the cytokinesis-block micronucleus test was performed. Results obtained from these experiments demonstrated a dose-related decrease in genotoxicity following co-treatment of mouse lymphoma cells with three doses of caffeinated instant coffee. Both pre-treatment and co-treatment showed significant antigenotoxic effects against MNNG. Caffeinated and decaffeinated instant coffee samples inhibited genotoxicity. There was no significant change in the antigenotoxic effect of caffeinated instant coffee after filtration using a 0.2 microm filter. Similar in vitro experiments demonstrated antigenotoxic effects against MNNG when boiled coffee was used instead of instant coffee. On the basis of the findings from the above preliminary experiments, further work was carried out to evaluate the possible protective effects of caffeinated instant coffee against MNNG-induced DNA damage, mutation and chromosomal damage. Results from three or five independent experiments demonstrated significant protective effects of caffeinated instant coffee against MNNG-induced DNA damage in the comet assay, mutation at the Tk locus and chromosomal damage in the cytokinesis-block micronucleus test.

Tomato and garlic by gavage modulate 7, 12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice

Chemoprotection by dietary agents is a promising strategy for cancer prevention. The aim of the present study was to evaluate the combined effect of tomato and garlic against 7,12-dimethylbenz- [a]anthracene (DMBA)-induced genetic damage and oxidative stress in 12-14-week-old male Swiss albino mice. The animals were randomized into experimental and control groups and divided into eight groups of five animals each. Group 1 animals were injected intraperitoneally with 35 mg/kg body weight DMBA suspended in peanut oil as a single dose. Groups 2-4 animals received tomato (500 mg/kg body weight), garlic (125 mg/kg body weight) and a combination of tomato and garlic for 5 days by gavage, respectively, followed by DMBA 1.5 h after the final feeding. The doses of tomato and garlic correspond to the average human daily consumption. Animals in groups 5, 6 and 7 received tomato alone, garlic alone and tomato + garlic combination, respectively, for 5 days. Group 8 animals received the same volume of water and served as control. The incidence of bone marrow micronuclei and the extent of lipid peroxidation and the concentrations of antioxidants glutathione, glutathione peroxidase and glutathione-S-transferase were measured in the liver, 48 h after DMBA exposure. Increased frequency of micronuclei and enhanced lipid peroxidation accompanied by compromised antioxidant defenses were observed in DMBA-treated animals. Although pretreatment with tomato or garlic significantly reduced the frequency of DMBA-induced bone marrow micronuclei, the combination of tomato and garlic exhibited more profound effect in inhibiting DMBA-induced genotoxicity and oxidative stress. We suggest that a broad spectrum of antimutagenic and anticlastogenic effects can be achieved through an effective combination of functional foods such as tomato and garlic.

Protective effects of a mixture of dietary agents against 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice.

We investigated the effects of pretreatment with tomato, garlic, and turmeric, alone and in combination, against 7,12-dimethylbenz[a]anthracene (DMBA)-induced genetic damage and oxidative stress in male Swiss mice. Measurement of the incidence of bone marrow micronuclei as well as the extent of lipid peroxidation and the status of the antioxidants reduced glutathione, glutathione peroxidase, and glutathione-S-transferase in the liver and erythrocytes were used as biomarkers of chemoprotection. In DMBA-treated animals, increased frequency of bone marrow micronuclei was accompanied by enhanced lipid peroxidation and antioxidant depletion. Pretreatment with tomato, garlic, and turmeric alone and a combination of these agents significantly reduced the frequencies of DMBA-induced bone marrow micronuclei as well as the extent of lipid peroxidation. These changes may be mediated by the antioxidant-enhancing effects of the dietary agents. The results of the present study suggest that a diet containing even low levels of different naturally occurring compounds is effective in exerting antigenotoxic effects by inhibiting DMBA-induced oxidative stress.

Protective effects of ethanolic neem leaf extract on N-methyl-N'-nitro-N-nitrosoguanidine-induced genotoxicity and oxidative stress in mice.

We evaluated the effects of pretreatment with ethanolic neem leaf extract on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced genotoxicity and oxidative stress in male Swiss albino mice. The frequency of micronuclei (MN), concentrations of lipid peroxides and the status of the antioxidants, reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were used as intermediate biomarkers of chemoprotection. Animals were divided into four groups of five animals each. Animals in group 1 were given MNNG (40 mg/kg body weight) by intragastric intubation. Animals in group 2 received intragastric administration of ethanolic neem leaf extract at a concentration of 200 mg/kg body weight for 5 days followed by MNNG 1.5 h after the final feeding. Group 3 animals received ethanolic neem leaf extract alone for five days. Group 4 received the same volume of normal saline and served as control. The animals were sacrificed by cervical dislocation 27 h after the carcinogen exposure. In MNNG-treated mice, enhanced lipid peroxidation with compromised antioxidant defences in the stomach, liver and erythrocytes was accompanied by increase in bone marrow micronuclei. Pretreatment with ethanolic neem leaf extract significantly reduced MNNG-induced micronuclei and lipid peroxides and enhanced GSH-dependent antioxidant activities. The results of the present study demonstrate that ethanolic neem leaf extract exerts protective effects against MNNG-induced genotoxicity and oxidative stress by augmenting host antioxidant defence mechanisms.

Protective effect of Spirulina fusiformis on chemical-induced genotoxicity in mice.

Spirulina fusiformis given by oral route to mice at doses of 250, 500 and 1000 mg kg(-1) significantly inhibit the genotoxicity induced by cisplatin and urethane. In addition, a significant reduction in the extent of lipid peroxidation with concomitant increase in the liver enzymatic (GPx, GST, SOD, CAT) and non-enzymatic (reduced glutathione) antioxidants were observed.

Dose-dependent protection by tomato against 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice.

This study was designed to investigate the protective role of pretreatment with graded doses of freshly prepared tomato paste against 7,12-dimethylbenz[a]anthracene (DMBA)-induced genetic damage and oxidative stress in male Swiss mice. The incidence of bone marrow micronuclei and the extent of hepatic lipid peroxidation and the antioxidants glutathione, glutathione peroxidase, and glutathione S-transferase were monitored. Three different concentrations (0.5, 1, and 2 g/kg body weight) of tomato paste were tested for their anticlastogenic effects against DMBA (35 mg/kg body weight). Increased frequency of micronuclei and enhanced lipid peroxidation accompanied by compromised antioxidant defenses were observed in DMBA-treated animals. Pretreatment with all three doses of tomato paste significantly reduced the frequencies of DMBA-induced micronuclei and oxidative stress. These findings demonstrate that administration of tomato paste protects against the clastogenic effects of DMBA by decreasing lipid peroxidation and enhancing the antioxidant status.

Effect of Spirulina fusiformis on cyclophosphamide and mitomycin-C induced genotoxicity and oxidative stress in mice.

Spirulina fusiformis was tested for its possible in vivo protective effects against cyclophosphamide (CP) and mitomycin-C (MMC) induced genotoxicity and oxidative stress in mice. Pre-treatment with S. fusiformis (250, 500 and 1000 mg kg(-1), p.o., daily for 5 days) significantly reduced the chromosomal damage and lipid peroxidation with concomitant changes in antioxidants and detoxification systems. All the three tested doses were effective in exerting a protective effect against CP and MMC.

Inhibition of genotoxicity by saffron (Crocus sativus L.) in mice.

Experiments were carried out to ascertain whether or not saffron (dried stigmas of Crocus sativus L.), a commonly used agent for flavoring and coloring food can exert modulatory effects on the in vivo genotoxicity of cisplatin (CIS), cyclophosphamide (CPH), mitomycin C (MMC) and urethane (URE). For this purpose, Swiss albino mice were pretreated for five consecutive days with three doses (20, 40 and 80 mg/kg body weight) of the aqueous extract of saffron. Genotoxic effects were assessed in the mouse bone marrow micronucleus test. The results obtained suggest that pretreatment with saffron can significantly inhibit the genotoxicity of CIS, CPH, MMC and URE. This inhibitory effect was not always dose-dependent. In addition, the hepatic glutathione S-transferase (GST) activity was assessed in the control and treated animals. No significant change in GST activity was observed after pretreatment with saffron alone. Treatment with the genotoxins alone significantly inhibited GST activity. Saffron pretreatment attenuated the inhibitory effects of the genotoxins on GST activity.

Anti-genotoxicity of trans-anethole and eugenol in mice.

The naturally occurring flavouring agents trans-anethole and eugenol were evaluated for antigenotoxic effects in mice. The test doses of trans-anethole (40-400 mg/kg body weight) and eugenol (50-500 mg/kg weight) were administered by gavage 2 and 20 h before the genotoxins were injected intraperitoneally. Anti-genotoxic effects were assessed in the mouse bone marrow micronucleus test. Pretreatment with trans-anethole and eugenol led to significant antigenotoxic effects against cyclophosphamide (CPH), procarbazine (PCB), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and urethane (URE). In addition, trans-anethole inhibited the genotoxicity of ethyl methane sulfonate (EMS). Both trans-anethole and eugenol exerted dose-related antigenotoxic effects against PCB and URE. There was no significant increase in genotoxicity when trans-anethole (40-400 mg/kg body weight) and eugenol (50-500 mg/kg body weight) were administered alone.

Milk of calcium cholelithiasis in children.

Milk of calcium bile is uncommon and occurs mainly in the adult population. The authors report on 2 children, each having a distinct clinical history and presentation, and each with milk of calcium bile/calculi possessing differing chemical composition and highly notable gross morphology. J Pediatr Surg 36:644-647.

Anti-genotoxicity and glutathione S-transferase activity in mice pretreated with caffeinated and decaffeinated coffee.

In vivo anti-genotoxic effects of caffeinated and decaffeinated instant coffee were compared in mice after pretreatment either by gavage for 10 consecutive days or in the drinking water for 2 weeks. Changes in hepatic sulfhydryl (-SH) content and glutathione S-transferase (GST) activity were evaluated in pretreated animals. Both caffeinated and decaffeinated instant coffee induced a moderate increase in -SH content and GST activity following pretreatment (with 70, 140 and 280 mg/kg body weight) by gavage for 10 days. This enhancement was not always dose dependent. The maximum effect on GST activity was observed at a dose of 140 mg/kg body weight/day. However, such an effect was not observed after administration of drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. Results of the bone marrow micronucleus test for evaluating genotoxic effects revealed that both caffeinated and decaffeinated instant coffee (140 mg/kg body weight/day) could exert significant anti-genotoxic effects against ip injected benzo[a]pyrene (BP), cyclophosphamide (CPH), 7,12-dimethylbenz[a]anthracene (DMBA), mitomycin C (MMC) and procarbazine (PCB) in animals pretreated by gavage. Anti-genotoxic effects against BP, DMBA and urethane (URE) were evaluated in animals that received drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. With the exception of the anti-genotoxic effect of decaffeinated coffee against DMBA, there was no significant change in genotoxicity after the above pretreatment. From this work, there is no evidence for any significant difference in the in vivo anti-genotoxicity of caffeinated and decaffeinated instant coffee.

Antigenotoxicity studies in Drosophila melanogaster.

The fruit fly Drosophila melangaster with its well developed array of genotoxicity test systems has been used in a number of studies on antigenotoxicity of various compounds and mixtures. In recent years, the newly developed Somatic Mutation and Recombination Tests (SMART) have mainly been employed. These one-generation tests make use of the wing or eye imaginal disc cells in larvae and have proven to be very efficient and sensitive. They are based on the principle that the loss of heterozygosity of suitable recessive markers can lead to the formation of mutant clones of cells that are then expressed as spots on the wings or eyes of the adult flies. We have employed the wing spot test with the two markers multiple wing hairs (mwh,3-0.3) and flare (flr,3-38.8). Three-day-old larvae, trans-heterozygous for these markers, are treated chronically or acutely by oral administration with the test compound(s) or complex mixtures. For antigenotoxicity studies, chronic co-treatments can be used, as well as separate pre-treatments with an antigenotoxic agent followed by a chronic treatment with a genotoxin. After eclosion, the wings of the adult flies are scored for the presence of single and twin spots. These spots can be due to different genotoxic events: either mitotic recombination or mutation (deletion, point mutation, specific types of translocation, etc.). The analysis of two different genotypes (one with structurally normal chromosomes, one with a multiply inverted balancer chromosome) allows for a quantitative determination of the recombinagenic activity of genotoxins. Results of two separate studies presented: (1) instant coffee has antirecombinagenic but not antimutagenic activity in the wing spot test; and (2) ascorbic acid and catechin are able to protect against in vivo nitrosation products of methyl urea in combination with sodium nitrite.