Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.     

Different effects of newly isolated saponins on the mutagenicity and cytotoxicity of the anticancer drugs mitomycin C and bleomycin in human lymphocytes

Aim of the present paper was to assess by using the in vitro micronucleus (MN) test in human lymphocytes the effect of two plant extracts isolated from Bupleurum fruticosum (saponins) on the clastogenicity and cytotoxicity of the anticancer drugs mitomycin C (MMC) and bleomycin (BLM). One saponin showed a dose-dependent MMC-induced mutagenesis inhibition together with co-genotoxic effect on BLM-treated cultures. The remaining saponin did not significantly alter MN induction of both chemotherapeutic agents whereas it enhanced BLM cytotoxicity.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Clastogenicity in vitro of the Na, K, Ca and Mg salts of saccharin; and of magnesium chloride; consideration of significance

The sodium, potassium, calcium and magnesium salts of saccharin, and magnesium chloride, have been shown to be clastogenic to Chinese hamster lung (CHL) fibroblasts in vitro, but only at elevated dose levels (8-16 mg/ml). Saccharin acid was inactive to the limits of its solubility (4 mg/ml). When the data are expressed in terms of ionic concentration, each salt showed a similar clastogenic potency. This suggests that ionic effects induced by these salts in the assay medium may be the critical determinant of the clastogenic effects seen, rather than that the saccharin moiety presents a genotoxic insult to the chromosomes of the cells. The metal-chelating agents EDTA and EGTA were non-clastogenic, but the disodium salt of EDTA showed weak activity prior to toxicity at 0.5 mg/ml. The absence of a clastogenic response for the salts of saccharin at dose levels lower than 4 mg/ml is discussed within the context of the threshold-dependent tumour-promoting activity of high dose levels of sodium saccharin to the bladder of male rats. The doubtful value of conducting in vitro clastogenicity studies at dose levels greater than 10(-2) M is discussed.     

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Protective effect of DCTN (trans-dehydrocrotonin) against induction of micronuclei and apoptosis by different mutagenic agents in vitro

The use of medicinal plants to combat diseases has increased in the last years despite the little information available with regard to the possible health risks they represent. The aim of the present study was to determine in vitro the possible clastogenic, apoptotic and cytotoxic effects of the active principle of Croton cajucara, trans-dehydrocrotonin (DCTN), and determine its protective effect against three mutagenic agents using the micronucleus test (MN) and apoptosis index in CHO-K1 cells. Three DNA damage inducing agents were utilized in the clastogenicity and anticlastogenicity tests (methylmethane sulfonate (MMS), mitomycin C (MMC) and doxorubicin (DXR); a negative control (PBS) and solvent control were also included. DCTN at concentrations of 400, 320, 240, 160 and 80microM did not show clastogenic activity in cultured CHO-K1 cells in the micronucleus test, did not induce apoptosis and showed negligible cytotoxicity in all cases. DCTN at concentrations of 240 and 400microM was tested for protective activity using three treatment protocols in relation to positive controls: pre-treatment, simultaneous treatment and post-treatment. The micronucleus test showed a protective effect for DCTN which varied among the different treatment protocols and with regard to the different DNA damage inducing agents. In the apoptosis test, DCTN was seen to have a protective effect under the following conditions: (I) at both concentrations in relation to MMS, in all three treatment protocols; (II) at both concentrations against damage caused by MMC with pre-treatment and at the higher concentration with simultaneous treatment; (III) at both concentrations against DXR with simultaneous treatment. Therefore, DCTN itself is not a clastogenic or cytotoxic substance, and does not induce apoptosis the in vitro system used. These results together with findings reported for DCTN in vivo, support the indication of this active principle at these concentrations for therapeutic use.     

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).     

Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.     

Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes.

The genotoxicity of methyl mercury chloride (MMC, 0-25 x 10(-6) M) and dimethyl mercury (DMM, 0-434 x 10(-6) M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 x 10(-6) M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 x 10(-6) M and 1.73 x 10(-6) M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more clastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.     

Chromosome damage by dothistromin in human peripheral blood lymphocyte cultures: a comparison with aflatoxin B1

The clastogenic potential of the pine tree fungal toxin dothistromin was studied by metaphase chromosome analysis of stimulated human peripheral blood lymphocytes exposed in vitro. The frequency of gaps, breaks, deletions and exchanges was scored in a series of cultures from 3 different donors. 50 cells were analysed for each dose level on coded slides. Testing was performed with and without added metabolic activation (as S9 mix) and aflatoxin B1 was used as a positive control in all experiments. Dothistromin caused a dose-dependent increase in the frequency of gaps and deletions which was not dependent on added metabolic activation. Even at high doses of dothistromin only a very small number of complex exchange-type aberrations were seen. This is in contrast to aflatoxin B1 where such aberrations were seen at low dose levels and especially in cultures to which S9 mix was added. High doses of dothistromin caused culture toxicity manifesting as haemolysis of the donor red blood cells and reduction of mitotic index. Culture toxicity occurred without a marked increase in aberration frequency. This toxicity may be masking any major potential for clastogenicity by dothistromin.     

Further investigations on the clastogenicity of paracetamol and acetylsalicylic acid in vitro

Paracetamol (PCM) and acetylsalicylic acid (ASA), both widely used analgesics, were tested for their clastogenicity in V79 cells in vitro. Rat liver S9 mix and primary rat hepatocytes (PRH) were used as external activation systems. ASA was found to be negative with and without activation system in concentrations up to 10(-2) M. In contrast PCM induced concentration-dependent chromosomal aberrations with and without activation system within the range of 3 x 10(-3) and 10(-2) M. The greatest effects were observed following continuous treatment with PRH activation and without external metabolization. Pulse treatments without external metabolization, with S9 mix and PRH were less effective. The clastogenic potency of PCM seems to be partly independent of metabolic activation. Although clastogenic effects in vitro were observed only in very high concentrations pharmacokinetic data and other published mutagenicity data indicate that there might be a risk for human use. Peak plasma levels of more than 10(-4) M have been reported (Forrest et al., 1982) and 2 groups of investigators (Kocisova et al., 1988; Hongslo et al., 1990) found PCM to be weakly clastogenic in human lymphocytes in vivo in the maximum human therapeutic dose range.     

Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms

Because sodium fluoride (NaF) is widely used for prevention of dental caries, pathobiological effects of NaF were investigated on human oral mucosal fibroblasts. The results showed that NaF was cytotoxic to oral mucosal fibroblasts at concentrations of 4 mmol/L or higher. Exposure of cells to NaF for 2 h also inhibited protein synthesis, cellular ATP level and functional mitochondrial activities in a dose-dependent manner. However, incubation of cells with NaF up to 12 mmol/L for 2 h depleted only 13% of cellular glutathione level. The IC50 of NaF on cellular ATP level was about 5.75 mmol/L. Preincubation of the cells with pyruvate and succinate did not protect cells from NaF-induced ATP depletion. At concentrations of 4 mmol/L, 8 mmol/L and 12 mmol/L, NaF inhibited 31%, 56% and 57% of mitochondrial functions, respectively, after 2 h incubation. No significant inhibition for NaF was found at concentrations lower than 2 mmol/L (40 ppm). These results indicate that NaF can be toxic to oral mucosal fibroblasts in vitro by its inhibition of protein synthesis, mitochondrial function and depletion of cellular ATP. Because of repeated and long-term usage of NaF, more detailed studies should be undertaken to understand its toxic effects in vitro and in vivo.     

Effect of pH on the activity and stability of clastogens in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells

The effect of the pH of the medium on the clastogenic activity of several direct-acting and indirect clastogens was evaluated in the in vitro chromosomal aberration test with Chinese hamster ovary K1 cells. Furthermore, the stability of the chemicals in the cell culture medium was measured by HPLC over the pH range of 5.0-11.0. The activity of the direct-acting clastogens mitomycin C (MMC), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and 4-nitroquinoline-1-oxide (4NQO) at various pH values depended on their stability. In the case of ENNG, its clastogenic activity decreased to about one-fifth at pH 9 but was about twice as high at acidic pH compared with that at pH 7.4. This is consistent with the observation that ENNG is unstable at basic pH; the residual content of ENNG was 0.5% of the initial amount in cell culture medium at pH 9.0 after a 2-h incubation. 4NQO was unstable at strongly basic pH (pH 10-11), and MMC was unstable at pH 5.0 and pH 11.0. The frequencies of chromosomal aberrations induced by MMC and ENNG were correspondingly decreased at these pH values. On the other hand, the clastogenicities of the indirect clastogens 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN), which require metabolic activation, were reduced at pH 10-11 and pH 5.8. The frequencies of chromosomal aberrations at these pHs were almost equal to negative control values. These chemicals were stable in the medium in the absence of S9 mix over the pH range of 5.0-11.0. Thus clastogenicity of indirect-acting clastogens is reduced under extreme pH conditions, probably because of the instability or nonformation of the active form. The present results indicate that the clastogenic activity of any compound will depend on its stability in the medium irrespective of its direct- or indirect-acting nature. In addition, some of the chemicals that are recognized as clastogens presumably might induce chromosomal aberrations by means of acidic pH itself. It is, therefore, important to take account of the pH of the treatment medium in evaluating the clastogenicity of chemicals.     

Clastogenic activity of sodium fluoride in great ape cells

Conflicting evidence has been reported concerning the mutagenicity of sodium fluoride (NaF), especially clastogenicity at concentrations of more than 1 mM. NaF is known to induce chromosome aberrations at these concentrations in human cells, but not in most rodent cells. We considered that such species-specific difference in chromosomal sensitivity would be derived from the phylogenetic distance between rodents and man. To clarify the role of interspecies differences, we investigated the chromosomal sensitivity to NaF in cell lines from various primates, which diverged into many species, including rodent-like prosimians and human-like great apes. The results showed that the clastogenicity of NaF was limited to human and great ape cells.     

Spontaneous and mitomycin-C induced sister-chromatid exchanges : Comparison of in vivo and in vitro systems

Frequencies of sister-chromatid exchanges (SCE) were measured in vitro in mouse fibroblasts and in vivo in mouse bone-marrow cells. SCE levels in these cell systems were measured in response to varying concentrations of bromodeoxyuridine (BrdU) and mitomycin-C (MMC). Although BrdU was found to induce SCE in both cellular systems, baseline SCE levels were 2- to 3-fold higher in vitro than in vivo. SCE induction was found to be a linear function of MMC concentration in vivo and in vitro; however the slope of the in vivo curve was 5-fold higher. The interaction of BrdU substituted DNA and MMC was examined by administering a fixed dose of MMC with increasing concentrations of BrdU. The induced SCE frequencies appeared to be additive. In addition to measuring drug-induced SCE, the BrdU differential staining technique allows concomitant measurement of the inhibition of cellular replication by the test drugs.     

Genotoxic effects of rotenone on cultured lymphocytes

Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle.     

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.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

Clastogenic effects of cis-diamminedichloroplatinum. I. Induction of chromosomal aberrations in somatic and germinal cells of mice

The clastogenicity of cisplatin, cis-diamminedichloroplatinum(II), an extensively used antitumor drug, has been studied employing (101/E1 X C3H/E1)F1 mice, aged 12-14 weeks. Chromosomal aberrations were assessed in mitotic divisions of bone marrow cells and differentiating spermatogonia. The drug was tested at 3 doses, 0.5, 1.0 and 2.5 mg/kg and 1.0, 2.5 and 5.0 mg/kg, respectively, for bone marrow and spermatogonia. Cisplatin had a clastogenic effect which was dose-dependent in both cell types. The frequencies of aberrant cells increased non-linearly in bone marrow and the dose-response relationship could be best described by a linear-quadratic equation. At the highest dose the affected cells carried multiple aberrations. An average of 2.7 aberrations per aberrant cell was observed 12 h after treatment of the mice with 2.5 mg/kg of cisplatin. In differentiating spermatogonia the dose response for aberrant cells could be described by a linear equation. The damage to the individual affected cell was less dramatic than in bone marrow, averaging 1.4 aberrations per damaged cell at the highest dose tested. Gaps were excluded from these considerations but they generally also showed a dose-related increase. A quantitative comparison of the clastogenic response to cisplatin was based on the dose-response relationships using 2 criteria, the doubling dose and the dose of unit increase (DUI). For both comparisons the general conclusion was that bone marrow cells were twice as sensitive as differentiating spermatogonia to the clastogenic action of cisplatin.