The effect of hyperthermia on micronucleus induction by mutagens in mice

We administered mitomycin C (0.5 mg/kg) intraperitoneally to hyperthermic-treated mice and examined the effect of hyperthermia on micronucleus induction. Hyperthermia enhanced micronucleus induction. The timing of chemical administration relative to the start of hyperthermic treatment (37 degrees C ambient temperature) influenced micronucleus frequency, and the effect was greatest 2 h after the start of hyperthermic treatment. But the hyperthermic treatment did not change the time course of micronucleus induction. In addition, we investigated the effect of hyperthermia on micronucleus induction by chemicals with different modes of action, i.e., alkylating agents (mitomycin C at 0.1-0.5 mg/kg, cyclophosphamide at 1.25-10 mg/kg), a spindle poison (colchicine at 0.05-1.0 mg/kg), and an antimetabolite (5-fluorouracil at 2.5-50 mg/kg). Hyperthermia enhanced only the clastogenicity of alkylating agents.     

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.     

Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes.

A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 micrograms/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

The effects of morphine and various narcotic antagonist type analgesics on body temperature in rats

ED50s were determined for morphine, nalorphine, butorphanol and pentazocine induced hyperthermia in rats. Morphine produced a significant hyperthermia with the doses of 5–160 mg.kg in rats. The peak hyperthermic effect was found 1 hr after 5–20 mg/kg doses of morphine. Nalorphine, butorphanol and pentazocine produced biphasic effects on rectal temperature. Initially they produced a dose-dependent hyperthermia and later hypothermia. In a comparison of the hyperthermic ED50's of morphine, nalorphine, butorphanol and pentazocine it was found that butorphanol is more active than the others (ED50s were 4.7, 4.3, 0.54 and 11.5 mg/kg respectively). The narcotic antagonist naloxone significantly inhibited both morphine and antagonist type analgesic induced hyperthermia. These results suggests that a different mechanism(s) is involved in the hyperthermic actions of antagonist type analgesics and agonist drugs.     

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 μg/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 μg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 μg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotne has been tested in the conventional mouse bone marrow assay. Single doses of 1 mg/kg bw or 2 mg/kg bw were given by oral intubations and bone marrow was sampled at 24 h (1 mg/kg) or at 6, 12 and 18 h after treatment (2 mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1 mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6 h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nocotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotine has been tested in the conventional mouse bone marrow assay. Single doses of 1mg/kg bw or 2mg/kg bw were given by oral intubations and bone marrow was sampled at 24h (1mg/kg) or at 6, 12 and 18 h after treatment (2mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nicotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Detection of micronuclei in peripheral blood of mitomycin C-treated mice using supravital staining with acridine orange.

The induction of micronuclei in peripheral blood from mitomycin C (MMC)-treated mice was examined using a supravital acridine orange staining method. Male ICR mice were intraperitoneally given MMC at a single dose of 0.25, 0.5, 1, or 2 mg/kg. Blood was sampled from the tail 24, 48, 72, and 96 h after treatment, and the frequency of micronucleated reticulocytes (MNRETs) was examined. The induction of MNRETs peaked at 48 h after treatment with MMC; there was a clear, dose-related increase in MNRETs. In a multiple-treatment study, mice were treated with 4 consecutive daily injections of MMC at a dose of 0.13, 0.25, 0.5, or 1 mg/kg. The frequency of MNRETs increased markedly 24 h after the second treatment as compared with the first treatment, and did not change significantly until 24 h after the fourth treatment. The frequency of MNRETs decreased to approximately control values 96 h after the last treatment. In addition, a slight but statistically significant increase in the number of micronucleated normochromatic erythrocytes in peripheral blood was detected by means of Giemsa staining 7 days after the last treatment. These results confirm the usefulness of the supravital acridine orange staining method to evaluate micronucleus induction in mouse peripheral blood.     

Effects of methylglyoxal bis(guanylhydrazone) on tumour and skin responses to hyperthermia in mice

Effects of methylglyoxal bis(guanylhydrazone) (MGBG) on tumour and skin responses to hyperthermia (42 degrees C) were examined in C3H mice. MGBG (50 mg/kg) was administered intraperitoneally to mice 4 hours before hyperthermic treatment. The tumour (FM3A) growth time was elongated by an amount dependent on the exposure time of treatment at 42 degrees C (60, 90 and 120 min). Pre-treatment of mice with MGBG (50 mg/kg, i.p.) apparently further lengthened the tumour growth time after treatment at 42 degrees C. No significant damage of foot skin was caused by 42 degrees C hyperthermia. Pre-treatment with MGBG did not make the foot skin susceptible to the heating. From these findings, it can be considered that MGBG or related less-toxic compounds may have a clinical advantage for the mild (42 degrees C) hyperthermic treatment in cancer therapy.     

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

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.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9–9.4 J/cm² of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Micronucleus test and bone-marrow chromosome analysis: A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations

The sensitivities of 2 cytogenetic tests, chromosome analysis and the micronucleus test, were compared by using mice exposed to the substances methyl methanesulfonate (MMS), mitomycin C (MC) and procarbazine (Natulan®). The lowest dose at which a significant effect could be observed in bone-marrow cells of mice was determined. Both test systems proved equally sensitive for MC and procarbazine. Doses as low as 0.16 mg of MC per kg and 3.12 mg of Natulan® per kg significantly increased both the aberration rates and the micronucleus rates above those of the controls. In contrast, after exposure to MMS, chromosomal aberrations were elevated above control levels at 5 mg/kg, and the micronucleus rate differed significantly from that of the controls after a dose of 10 mg/kg. With the present protocol and sample size one can conclude that the micronucleus test is generally comparable in sensitivity to the chromosome analysis. However, the MMS data indicate that there might be chemicals for which the resolution of the chromosome analysis is higher.

When the mutagens were given in 2 single i.p. injections separated by 24 h, the polychromatic erythrocytes were analyzed for the presence of micronuclei 6 or 24 h after the second injection. The double treatment did not increase the micronucleus rates above the single-treatment results at either sampling interval.     

Alteration of poly(ADP-ribose) metabolism by hyperthermia

The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.     

Hyperthermic inactivation of diploid yeast and the interaction of damage caused by hyperthermia and ionizing radiation.

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.     

Beta-carotene as an inhibitor of benzo(a)pyrene and mitomycin C induced chromosomal breaks in the bone marrow of mice

Female mice of hybrid strain B6C3F1, 8-10 weeks old, were fed on powdered food with or without beta-carotene (100 mg/kg food). After 1 week of these diets, some of each group of mice were injected i.p. with either benzo(a)pyrene (150 mg/kg) in dimethyl sulfoxide, or mitomycin C (1 mg/kg) in distilled water. In the course of separate experiments, bone marrow samples were collected at various intervals after injection for analysis in the in vivo bone marrow micronucleus assay. At the time at which the maximum induction was observed, which coincided between experiments, the frequency of micronuclei induced by benzo(a)pyrene was reduced by 41-61% and that induced by mitomycin C was reduced by 44-71% in the presence of beta-carotene. Beta-carotene is widely distributed in plant material such as carrots and green leafy vegetables and, as such, is a component of the human diet. Our results suggest that beta-carotene provides significant protection against the genotoxicity of benzo(a)pyrene and mitomycin C.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.