Genotoxic,Cytotoxic, Antigenotoxic,and Anticytotoxic Effects of Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow Micronucleus Test

Chalcones present several biological activities and sulfonamide chalcone derivatives have shown important biological applications, including antitumor activity. In this study, genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of the sulfonamide chalcone N-{4-[3-(4-nitrophenyl)prop-2-enoyl]phenyl} benzenesulfonamide (CPN) were assessed using the Salmonella typhimurium reverse mutation test (Ames test) and the mouse bone marrow micronucleus test. The results showed that CPN caused a small increase in the number of histidine revertant colonies in S. typhimurium strains TA98 and TA100, but not statistically significant (p > 0.05). The antimutagenicity test showed that CPN significantly decreased the number of His⁺ revertants in strain TA98 at all doses tested (p < 0.05), whereas in strain TA100 this occurred only at doses higher than 50 μg/plate (p < 0.05). The results of the micronucleus test indicated that CPN significantly increased the frequency of micronucleated polychromatic erythrocytes (MNPCE) at 24 h and 48 h, revealing a genotoxic effect of this compound. Also, a significant decrease in polychromatic/normochromatic erythrocyte ratio (PCE/NCE) was observed at the higher doses of CPN at 24 h and 48 h (p < 0.05), indicating its cytotoxic action. CPN co-administered with mitomycin C (MMC) significantly decreased the frequency of MNPCE at almost all doses tested at 24 h (p < 0.05), showing its antigenotoxic activity, and also presented a small decrease in MNPCE at 48 h (p > 0.05). Additionally, CPN co-administered with MMC significantly increased PCE/NCE ratio at all doses tested, demonstrating its anticytotoxic effect. In summary, CPN presented genotoxic, cytotoxic, antigenotoxic, and anticytotoxic properties.     

Absence of antimutagenicity of Cochlospermum regium (Mart. and Schr.) Pilger 1924 by micronucleus test in mice.

Cochlospermum regium (Mart. and Schr.) Pilger, popularly known as "algod?ozinho do campo", is a medicinal plant that grows in the Cerrado of Brazil. This plant has been used in traditional medicine against various diseases such as leucorrhoea, gastritis and ulcers. It has also been effective in treating skin problems like pimples, boils and blotches. In the present study, the in vivo antimutagenicity of aqueous extract of C. regium was evaluated. The Micronucleus Test was performed in polychromatic erythrocytes from Swiss male mice treated with one of the four doses of extract of the plant (19, 38, 76 and 114 mg.kg(-1) body weight), administered by intraperitonial injection (i.p.) simultaneously with cyclophosphamide (24 mg.kg(-1) b.w.) or mitomycin C (4 mg.kg(-1) b.w.). The cytotoxicity was evaluated by polychromatic and normochromatic erythrocytes ratio (PCE/NCE). The results showed no significant reduction of the micronucleated polychromatic erythrocytes frequency (P > 0.05). In conclusion, the data indicate that C. regium roots aqueous extract, for the conditions used, did not exhibit the antimutagenic effect.     

The effect of diphenylhydantoin on the frequency of micronuclei in bone-marrow polychromatic erythrocytes of mice

Using the micronucleus test to evaluate the mutagenic effect of 5,5-diphenylhydantoin (DPH) on bone marrow polychromatic erythrocytes, male Balb-C mice were treated with the drug in single and multiple injection tests. A significant increase in the frequency of micronucleated polychromatic erythrocytes (MPE), P less than 0.05, was found when the mice received a single injection of DPH at doses of 0.5 and 1.0 mg/kg, and this frequency did not increase at higher doses. When mice were treated 3 times, at 24-h intervals, with 1.0 mg/kg of DPH, a significant increase in MPE was also observed (P less than 0.05) but this was lower than when they received a single injection of the same dose. A cytotoxic effect of NaOH, 0.1 N, which was used as solvent, was also observed either when alone or when DPH (1.0 mg/kg) was injected 3 times. This effect was comparable to the one produced by mitomycin C (MMC) at a dose of 0.5 mg/kg.     

Evaluation of the genotoxic and antigenotoxic potential of Melissa officinalis in mice

Melissa officinalis (L.) (Lamiaceae), a plant known as the lemon balm, is native to the east Mediterranean region and west Asia. Also found in tropical countries, such as Brazil, where it is popularly known as "erva-cidreira" or "melissa", it is widely used in aqueous- or alcoholic-extract form in the treatment of various disorders. The aim was to investigate in vivo its antigenotoxicity and antimutagenicity, as well as its genotoxic/mutagenic potential through comet and micronucleus assaying. CF-1 male mice were treated with ethanolic (Mo-EE) (250 or 500 mg/kg) or aqueous (Mo-AE) (100 mg/kg) solutions of an M. officinalis extract for 2 weeks, prior to treatment with saline or Methyl methanesulfonate (MMS) doses by intraperitoneal injection. Irrespective of the doses, no genotoxic or mutagenic effects were observed in blood and bone-marrow samples. Although Mo-EE exerted an antigenotoxic effect on the blood cells of mice treated with the alkylating agent (MMS) in all the doses, this was not so with Mo-AE. Micronucleus testing revealed the protector effect of Mo-EE, but only when administered at the highest dose. The implication that an ethanolic extract of M. officinalis has antigenotoxic/antimutagenic properties is an indication of its medicinal relevance.     

Induction of micronuclei in mouse bone-marrow erythrocytes in association with hypothermia after administration of the sigma receptor ligand E-5842

Oral administration of E-5842, a new sigma1 receptor ligand being developed as an antipsychotic drug, to male mice at single doses of 50, 100, 200 and 400 mg/kg produced marked and sustained decreases in rectal temperature. Both the intensity and the duration of the hypothermic effect increased with dose. Maximum decreases from the mean pre-administration temperature (36.2 degrees C) ranged from 7.5 to 12.9 degrees C for animals receiving 50 and 400 mg/kg doses, respectively. Examination of bone-marrow smears obtained 24, 48 and 72 h after administration revealed a slight but statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) at the 48 h sampling for animals receiving the 200 mg/kg dose. These animals showed decreases from pre-administration temperature of approximately 12 degrees C, with recovery being observed 24 h after administration. When the hypothermic effect of E-5842 administration was avoided by housing treated animals under conditions of increased environmental temperature (30 degrees C) for 24 h, MNPCE frequency reverted to vehicle control values. Further, in E-5842-treated animals with an increased MNPCE frequency there was a shift in the distribution of the relative areas of micronuclei in MNPCE towards higher values. In addition, there was a statistically significant increase (p < 0.001) in the number of relatively large micronuclei (micronucleus diameter > or = 1/4 cytoplasm diameter) similar to that produced by administration of the mitotic spindle inhibitor colchicine (1 mg/kg), suggesting disturbance of mitotic apparatus as the possible underlying mechanism. The results suggest that the slight increase in MNPCE frequency observed 48 h after administration of a 200 mg/kg dose of E-5842 is due to a hypothermic effect and not to a direct effect of E-5842 on DNA.     

Efficiency of Coleus aromaticus extract in modifying cyclophosphamide and mitomycin-C induced clastogenicity in mouse bone marrow cells

The anticlastogenic potency of the ethanolic extract of a medicinal plant, C. aromaticus was investigated by taking bone marrow chromosomal aberration assay and micronucleus (MN) test as the test parameters. Swiss albino mice were fed orally with different doses (10,15, 25, 50 and 100 mg/kg body weight) of ethanolic extract for 7 days and on the 7th day, two doses each of anticancer drugs cyclophosphamide (CP; 25 and 50 mg/kg body weight) and mitomycin-C (MMC; 4 and 8 mg/kg body weight) were injected, ip, to different groups of animals. Bone marrow MN preparations were made at 24 and 48 hr time intervals. Coleus extract reduced CP and MMC induced MN and lower doses of the extract were found to be more effective than higher doses. The effective doses of extract in MN test were selected to study the anticlastogenic effects against CP (25 and 50 mg/kg body weight) and MMC (2 and 4 mg/kg body weight) induced chromosomal aberrations. The results indicate the protective effect of C. aromaticus against CP and MMC induced cytogenetic damage.     

The genotoxic and cytotoxic effects of nicotine in the mouse bone marrow

The objective of the present study was to investigate the potential of nicotine to induce micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of male and female mice. Cyclophosphamide at 40mg/kg was used as positive control clastogen. Single doses of 4, 8 or 16mg/kg nicotine were given via oral intubation and bone marrow was sampled at 18, 24, 30, 36 and 48h after treatment. Cyclophosphamide yielded the expected positive results. Despite the evident signs of acute toxicity shown by the animals, mainly at the 8 and 16mg/kg doses of nicotine, and the reduction in the % PCE, the results show that the MNPCE frequency in male and female mice was not affected by treatment with any of the selected doses of nicotine, in either of the sampling times 18 or 24h. However, at 30 and 36h after treatment, the MNPCE showed significant increases in both genders after doses of 8 and 16mg/kg. A sex-dependent response was recorded, with males having more MNPCE than females after treatment with 8 or 16mg/kg nicotine and sampling at 30h. However, at 36h more MNPCE were induced in females than in males, suggesting different degrees of dose interaction in the sexes under the conditions of the assay. The response was directly correlated with bone-marrow toxicity, as greater bone-marrow suppression was noted in females than in males when 36h samples were examined. By 48h recovery was observed even though the cytotoxicity was high. These findings suggest that nicotine at high doses and after prolonged time intervals is genotoxic and cytotoxic for mouse bone marrow.     

In vivo cytogenetic effects of a commercially formulated mixture of cypermethrin and quinalphos in mice

In vivo cytogenetic effects of commercially formulated cypermethrin (CYP, synthetic pyrethroid insecticide) and/or quinalphos (QUI, organophosphate insecticide), generally used in combination, were examined through chromosomal aberrations (CA) and micronucleus test (MT) in mice. Male mice were orally gavaged to a single dose of CYP/QUI commercial mixture (22, 44 or 67 mg/kg b.wt.) for 24h (CA) or 48 h (MT). Based on the concentrations of active ingredients of CYP and QUI present in the test doses of CYP/QUI mixture, mice were orally exposed to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. For reference, a group of five mice was intraperitoneally administered to cyclophosphamide (20 or 50 mg/kg) or orally gavaged to peanut oil for vehicle control. Exposure of CYP/QUI mixture inhibited the mitotic index (MI) and induced CA in a dose-dependent manner at 24 h; however, significant (p<0.01 or 0.001) frequencies of CA were observed at 44 mg/kg onwards, whereas inhibition of MI at 67 mg/kg. Independent exposure of QUI at 8.8 mg/kg onwards also significantly (p<0.01 or 0.001) inhibited MI and induced CA, whereas CYP at 2 mg/kg (highest concentration in CYP/QUI mixture) inhibited MI significantly but failed to induce CA. Chromatid breaks and fragments found to be frequent aberrations in all the test groups. Treatment of CYP/QUI mixture also induced micronucleus formation dose-dependently at 48 h, yet statistically significant (p<0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE) were observed at 44 mg/kg onwards. QUI (8.8 and 13.4 mg/kg) alone also induced significant frequencies of MNPCE, whereas frequencies of MNPCE observed with the CYP even at 2 mg/kg were comparable to that of vehicle control. Present findings indicate the genotoxicity potential of CYP/QUI mixture and suggest that the simultaneous presence of the toxic doses of CYP and QUI can lead to synergistic genotoxicity in mice and may pose mutagenic risk in human beings.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Activity of a nitroalkene derivative, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene, in the Salmonella/microsome assay and the mouse bone marrow micronucleus test

Mutagenicity of a substituted nitroalkene, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene (BNF) was tested in the Salmonella/microsome assay using the strains TA 98, TA 100 and TA 100NR (nitroreductase deficient). BNF was a direct mutagen in TA 98 and TA 100; the response was lowered when exogenous metabolic activation (S9) was used. A further decrease in mutagenicity was observed in strain TA 100NR, as compared to the parental TA 100, which showed the involvement of nitroreduction in the overall response elicited by BNF. The micronucleus assay was carried out in Swiss male mice which were given a single i.p. dose of 10–20 mg/kg of BNF dissolved in peanut oil, bone marrow being sampled 24 and 48 h later. The micronucleated polychromatic erythrocyte counts (MNPCE) showed a weak response in the dose range of 10–17.5 mg/kg at the second sampling (48 h) and a significant rise for 20 mg/kg at 24 and 48 h.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Effect of glutathione on mitomycin C-induced micronuclei in bone marrow erythrocytes of Swiss albino mice

The antimutagenic potential of glutathione (GSH) on mitomycin C (MMC)-induced micronuclei was evaluated in Swiss albino mice using the in vivo bone marrow micronucleus test. Six groups of animals were maintained simultaneously. The first group received distilled water only, the second group of animals received 2 mg/kg MMC and the third group was administered 4 doses of GSH, i.e., 20, 40, 80 and 160 mg/kg. The fourth group of animals received GSH and MMC simultaneously. The fifth and sixth groups received a cumulative dose of GSH followed by MMC after 24 h. The fifth group of animals were killed 6 h after the administration of MMC, while the sixth group were killed 24 h after the administration of MMC. The results clearly show a statistically significant increase in micronuclei in MMC-treated animals and also in animals that received GSH followed by MMC. However, there was a decrease in micronuclei in animals that received GSH and MMC simultaneously. The results clearly indicate that GSH exhibits an antimutagenic property in the presence of MMC. It is also observed the treatment with GSH prior to MMC does have some protective effect.     

Preliminary evaluation of anti-inflammatory and anti-arthritic activity of S. lappa, A. speciosa and A. aspera.

Saussurea lappa, Argyreia speciosa and Achyranthes aspera are well known Indian medicinal plants used in the indigenous systems of medicine for the treatment of inflammatory conditions. The ethanolic extracts of the plants at the doses of 50, 100 and 200 mg/kg, p.o. were screened for their effect on acute and chronic inflammation induced in mice and rats. S. lappa and A. speciosa were found to significantly inhibit paw edema induced by carrageenan and Freund's complete adjuvant and to prevent accumulation of inflammatory cells in carrageenan-induced peritonitis at doses of 50-200 mg/kg. A. aspera inhibited these inflammatory responses at doses of 100-200 mg/kg. The studies reveal that the ethanolic extracts of S. lappa, A. speciosa and A. aspera possess anti-inflammatory and anti-arthritic activity and support the rationale behind the traditional use of these plants in inflammatory conditions.     

The mutagenic and antimutagenic effects of Ecballium elaterium fruit juice in human peripheral lymphocytes

The aim of this study was to investigate the mutagenic and antimutagenic effects of Ecballium elaterium (EE) fruit juice, which has an anti-inflammatory effect, using in vitro human peripheral lymphocytes. To investigate the mutagenic effects of the EE fruit juice, human peripheral lymphocytes were treated with three doses (18, 36, and 72 μl/l) of fruit juice alone for 24 and 48 h. For investigating the antimutagenic effects of the EE fruit juice, the human lymphocytes were also treated with the mixture of the fruit juice and 0.25 μg/ml MMC. The EE fruit juice induced the percentage of total CA when used alone (especially the percentage of structural CA than the percentage of the numerical CA) and synergically induced the percentage of total CA when used as a mixture with MMC. The EE fruit juice did not affect the SCE frequency for 24 and 48 h treatment time. In contrast, EE and MMC as a mixture sinergically induced the SCE frequency at the highest concentration for 48 h treatment time only. EE alone did not decrease the RI while it decreased the MI in a dose-dependent manner. EE and MMC as a mixture have a higher cytotoxic effect than the cytotoxic effects of EE alone. As a result, it can be concluded that EE had no antimutagenic effect while EE had a mutagenic and a cytotoxic effect in human peripheral lymphocytes. This article was submitted by the authors in English.     

Micronucleus formation in cultured fetal liver blood cells using mitomycin C.

Mouse fetal-liver blood cells were cultured and used to investigate micronucleus formation after exposure to mitomycin C (MMC). The isolated fetal cells were incubated in a medium supplemented with erythropoietin (EPO), and the frequency of micronuclei formation was detected in polychromatic erythrocytes (PCE). The effects of four variables were investigated: (1) MMC exposure dose, (2) MMC exposure time, (3) incubation time, and (4) EPO concentration. PCE were formed by proliferation and differentiation of the erythroid cells in culture. Micronucleated PCE (MNPCE) were observed in a dose-dependent manner after exposing the cultured cells with up to 1.0 microgram/ml MMC. The optimum time of MMC exposure and post-exposure incubation was 3 h and 48 h, respectively, and the optimum EPO concentration was 0.25 U/ml. Mouse fetal-liver PCE are sensitive primordial cell targets that can be obtained in relatively large numbers from a single pregnant animal. The procedure is relatively simple and potentially useful in detecting mutagens and carcinogens capable of causing chromosomal damage.     

A comparison of intraperitoneal injection and oral gavage in the micronucleus test with mitomycin C in mice

Intraperitoneal (i.p.) injection and oral (p.o.) gavage were evaluated in the mouse micronucleus test with mitomycin C (MMC). The tests were carried out in 2 laboratories with the MS/Ae and CD-1 mouse strains. On the basis of a small-scale acute toxicity study and a pilot experiment, the full-scale micronucleus test was performed with a 24-h sampling time at doses of 1, 2, 4, and 8 mg/kg for both treatment routes. In both strains, a clear positive dose-response relation was shown by both routes. Although the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was higher with i.p. on a mg/kg basis, this tendency was reversed when dose was expressed as a percentage of the LD50.     

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.     

Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis

Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.     

In vivo studies on genotoxicity of pure and commercial linuron

The ureic herbicide linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and d-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: `comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and d-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel electrophoresis technique (`comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.