Tissue-specific clastogenic effects of chromium and selenium salts in vivo

The clastogenic effects of inorganic compounds of chromium (K2Cr2O7) and selenium (Na2SeO3) on the chromosomes of rat lymphocytes and bone marrow have been investigated. In vitro exposure of rat lymphocytes to K2Cr2O7 gave highly significant and dose-related increases in abnormal metaphases at 6 concentrations from 7 X 10(-6) M to 3.2 X 10(-5) M. Similar in vitro exposure of lymphocytes to Na2SeO3 showed that it was clastogenic at concentrations of 7.5 X 10(-6) M, 1 X 10(-5) M and 2.5 X 10(-5) M. However, with in vivo exposures of K2Cr2O7 (i.p. and i.v.) it was only possible to demonstrate clastogenicity in lymphocytes at sublethal concentrations (36 mg/kg X 2 i.v.) and then only if the results were tested against all controls combined (1900 metaphases, 19 animals). On the other hand, very highly significant clastogenic effects were obtained in bone marrow cells exposed in vivo to K2Cr2O7 at 21 mg/kg i.p. and 12, 18, 24 and 36 mg/kg i.v. In vivo exposure to Na2SeO3, with concentrations up to 6 mg/kg X 2 i.v., caused no significant increase in abnormal metaphases in lymphocytes but 5 and 6 mg/kg X 2 i.v. caused a significant increase in abnormal metaphases in bone marrow. These results suggest that K2Cr2O7 and Na2SeO3 are acting as 'S' dependent chemicals. Although not directly comparable, they are compatible with the warnings given by other authors both on the detection of aberrations in lymphocytes after chronic exposure in man and on short-term testing of lymphocytes at low doses related to human exposure.     

Clastogenic activity of urethane in mice.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice

Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57Bl or BDF1 (C57Bl x DBA2) mice with caffeine (1 or 3 x 50 mg/kg and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57Bl and BDF1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57Bl compared to BDF1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57Bl mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57Bl and BDF1 mice but not in fetal mouse livers.     

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

In vivo effect of selenium on the mutagenic activity of aflatoxin B1

Experiments were carried out to verify the effect of selenium on the mutagenic activity of AFB1. After 14 days of selenium administration to experimental animals (Chinese hamsters, Cricetulus griseus) in the form of 2 ppm Na2SeO3 solution available ad libitum the incidence of chromosomal aberrations in bone marrow cells due to a single p.o. administration of 5 mg AFB1 per 1 kg body weight was significantly reduced. The incidence of chromosomal aberrations was monitored till day 32 after AFB1 administration. A significant decrease in the frequency of aberrant cells, breaks and gaps was observed at almost any time during the investigation. 2 ppm Na2SeO3 solution itself did not enhance the frequency of chromosomal aberrations. The mechanism of the protective effect of selenium vis-a-vis the mutagenic and carcinogenic action of AFB1 remains obscure.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures.

Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17beta-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24h with Vitamin E (Vit E), and (4) pre-treatment for 4h with 17beta-estradiol (17beta-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2-20 mg/kg bw). These doses corresponded to 0.4-4% of the LD50 in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 x 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 x 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a 'hit and go' manner. Furthermore, pre-treatment of animals with 17beta-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.     

Sister-chromatid exchange induction by sodium selenite: reduced glutathione converts Na2SeO3 to its SCE-inducing form

Sodium selenite (Na2SeO3) is an anticarcinogenic/antimutagenic agent that exhibits carcinogenic/mutagenic properties in some short-term test systems used for the detection of DNA-damaging agents. One such test system is sister-chromatid exchange (SCE) induction. Na2SeO3 induces SCEs only if red blood cells (RBCs) are present to 'activate' it to its SCE-inducing form. Here, the ability of reduced glutathione, a major component of RBCs, to serve as an RBC substitute in the activation of Na2SeO3 was determined. Reduced glutathione (10(-4) and 10(-3) M) was shown to be as capable as RBCs in activating Na2SeO3 (7.95 X 10(-6) M) to its SCE-inducing form. These data suggest strongly that the pathway normally utilized by RBCs in the metabolism of Na2SeO3 is the same as that in which Na2SeO3 is converted to its SCE-inducing form.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Municipal sludge leachate-induced genotoxicity in mice--a subacute study

Inappropriate disposal of municipal sludge (MS) results in the leaching of toxic metals and organic chemicals, which can contaminate the surface and ground water leading to the serious health hazards. In this study, the genotoxic potential of the leachate prepared from MS sample was examined in mouse bone marrow cells through chromosomal aberrations (CA), micronucleus test (MT) and comet assay. Analysis of metals and physicochemical parameters of the leachate was also carried out to correlate the genotoxic results. The dried sludge showed high concentrations of heavy metals, viz. Cr, Cu, Pb and Ni. However, in 10% leachate, concentrations of these metals were manifold lower than that of obtained in dried sludge. Male mice orally gavaged to leachates (0.1-0.4 ml/mouse/day) for 15 days revealed significant (P<0.01, P<0.001) inhibition of mitotic index (MI) and induction of chromatid/chromosome fragments and breaks in all the treatment groups. The effect was observed to be dose-dependent. Treatment of mice with leachates also induced significant (P<0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE). The results of comet assay revealed a statistically significant (P<0.05 and <0.01) DNA damage in bone marrow cells exposed to 0.2-0.4 ml/mouse/day. Findings of the present study indicate that the constant exposure of MS leachate can cause genotoxic effects in mammals and suggest risks in human population.     

Microcalorimetric studies of the action of Na2SeO3 on the growth of Halobacterium halobium R1

The effect of Na₂SeO₃ on the growth of Halobacterium halobium R1 was investigated by means of microcalorimetry at 37°C. The biological response to toxicants is observed as the inhibition of the rate constant of growth of living cells. A low concentration of Na₂SeO₃ stimulated the growth of H. halobium R1, and a high concentration of Na₂SeO₃ inhibited the growth of H. halobium R1. Toxicity may be expressed as the half-inhibition concentration (IC₅₀). The rate constants of growth (k) and the concentrations of Na₂SeO₃ (c) shows a linear relationship: k=1.790 × 10⁻⁶ − 2.27 × 10⁻³ c. The value of IC₅₀ obtained from the accompanying figure of I-c is 679 μg/mL.     

Toxic and cytogenetic effects induced in Allium cepa with low concenrations of Cd and 232th

232Th (7.76 x 10(-7) M) and Cd (0.89 x 10(-8) M) in concentrations which do not exceed officially prescribed standards when entering with water do not increase the frequency of chromosome aberrations in comparison with the control. Such concentrations do not cause toxic effects in plants on the levels of tissues and of the whole organism but they do display their activity on the cell level damaging division spindle. Dependence "cadmium concentration-effect" is not linear for any type of cytogenetical damages. At the concentration of cadmium 0.89 x 10(-7) M its influence on formation of division spindle is weakened and the frequency of chromosome aberrations is reducing in comparison with the control and with the effects induced at lower concentrations of cadmium in solution (0.89 x 10(-8) M). Cadmium in high concentration (5.34 x 10(-5) M) causes significant toxic and mutagenic effects.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

DNA-repair kinetics and the sensitivity of cells to X-ray-induced chromosome aberrations: a mouse myeloid leukemia cell line and normal mouse bone marrow cells

The frequency of X-ray-induced chromosome aberrations in G1 ML-1 mouse myeloid leukemia cells and normal mouse bone marrow cells increased with post-irradiation incubation with the DNA-repair resynthesis inhibitor 1-beta-D-arabinofuranosylcytosine (araC), but the frequency of aberrations in the leukemic cells increased with quite a different time response compared to the normal cells. Irradiated normal mouse bone marrow cells had a rapid increase in the frequency of chromosome exchanges and deletions with increasing araC incubation time, for example, an increase was observed with 0.5 h araC incubation. In contrast, the ML-1 cells did not have a significant increase in aberrations until 1-2 h post-irradiation incubation with araC. These results suggest that the ML-1 cells, per unit time, initially undergo less repair of the X-ray-induced DNA damage that can be converted into chromosome aberrations. We previously showed that the ML-1 cells have a higher frequency of X-ray-induced chromosome aberrations compared to normal cells and the results presented here indicate that a slower rate of repair resynthesis is contributing to the increased sensitivity of the ML-1 cells.     

Sodium selenite reduces hemoglobin-induced venular leakage in the rat mesentery

Modified Hbs are being developed as "blood substitutes," but intravascular injection of diaspirin cross-linked Hb (DBBF-Hb) can produce venular leakage. Hb toxicity may arise from reactive oxygen species, so the antioxidant sodium selenite (Na(2)SeO(3)) was used in an attempt to reduce leak formation. In anesthetized Sprague-Dawley rats, one-half of which received 2 x 10(-6) g/ml Na(2)SeO(3) in their drinking water for 3 wk, the mesenteric microvasculature was perfused with 2 mg/ml DBBF-Hb (N = 8) for 10 min. Controls (N = 7) received saline. This was followed by perfusion with FITC-albumin for 3 min, fixation, and microscopic examination. In rats given DBBF-Hb, Na(2)SeO(3) significantly reduced leak number, leak area, and mast cell degranulation. Venular leakage was also reduced in rats that only received Na(2)SeO(3) locally during DBBF-Hb perfusion. However, Na(2)SeO(3) did not affect animals receiving cyanomet-DBBF-Hb instead of DBBF-Hb and significantly increased leak number and mast cell degranulation in animals receiving saline. In vitro, Na(2)SeO(3) reduced the oxidation rate of DBBF-Hb while in the presence of oxidants. These results suggest that Na(2)SeO(3) reduces DBBF-Hb-induced microvascular leakage partly by retarding the oxidation of its heme iron.     

Evaluation of the mutagenic effect of the new fungicide trimorphamide

The new Czechoslovak fungicide trimorphamide was tested for its mutagenic activity. To evaluate the potential mutagenic effects on Drosophila, trimorphamide at 0.5, 1.0, 5.0, 10.0% was administered into the cultivation medium, and the sex-linked recessive lethal mutation detection test and the chromosome nondisjunction test were used. After administration of trimorphamide to mice at 60, 150 and 300 mg . kg-1 b.w. perorally, and 30, 70 and 150 mg . kg-1 b.w. intraperitoneally in single and repeated (5X) doses, a cytogenetic analysis of chromosomal aberrations in bone-marrow cells was performed. The cytogenetic analysis of human peripheral lymphocytes for chromosomal aberrations in vitro was performed 24 h after trimorphamide had been applied into the culture in concentrations 19.1 X 10(-3), 19.1 X 10(-4) and 19.1 X 10(-5) M. Under our testing conditions the trimorphamide concentrations used did not show any mutagenic effect upon Drosophila, compared with the controls. Also, under the conditions of the cytogenetic analysis, no significant increase in the frequency of chromosomal abnormalities in mouse bone marrow or in human peripheral lymphocyte was observed compared with the group of controls.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

MNNG-induced mutations in the adult gill and hepatopancreas and in embryos of rpsL transgenic zebrafish

To evaluate the feasibility of a mutagenicity assay using adult rpsL transgenic zebrafish, 4- to 8-month-old females were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0, 15 or 30 mg/L in a water bath for 2 h). At 2 weeks after exposure, MNNG showed a concentration-dependent significant increase in mutant frequency (MF) of 8 x 10(-5), 18 x 10(-5), and 51 x 10(-5), respectively, in the gill. DNA sequencing revealed that 60-74% of the induced mutations were G:C to A:T transitions, consistent with the known mutagenic effects of MNNG. A marginal but significant increase in MF was observed in the hepatopancreas only in the group exposed to 30 mg/L, with the induction of some G:C to A:T transitions. A time-course of the appearance of mutations was determined in fish treated with 15 mg/L MNNG. In both, the gill and hepatopancreas, a higher MF was observed at 3 weeks than at 2 weeks, suggesting that an expression time of at least 3 weeks is preferable for the assay. When embryos (29 h post-fertilization) were exposed to MNNG (0, 50, and 150 mg/L) for 1 h, MFs increased significantly with an increase in the concentration of MNNG (5 x 10(-5), 40 x 10(-5), and 144 x 10(-5), respectively) at 3 days after exposure. G:C to A:T transitions were the predominant mutations, and these occurred at the same sites in the rpsL gene as in adult tissues. Thus, MNNG induces typical mutations in the gill and hepatopancreas of adult fish, and in embryos, suggesting that the rpsL zebrafish is a useful tool for monitoring genotoxicity caused by water-borne mutagens.     

Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.     

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17β-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24 h with Vitamin E (Vit E), and (4) pre-treatment for 4 h with 17β-estradiol (17β-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2–20 mg/kg bw). These doses corresponded to 0.4–4% of the LD₅₀ in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 × 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 × 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a ‘hit and go’ manner. Furthermore, pre-treatment of animals with 17β-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.