Environmental hazard in the aetiology of somatic and germ cell aneuploidy.

Sources of environmental exposures to potentially aneugenic agents are many and include occupational and therapeutic exposures, and exposures associated with lifestyle habits. In this present study, some of these agents and exposure scenarios are discussed that involve potentially large population targets and/or seem to affect chromosome segregation by previously unsuspected mechanisms: metals, possibly acting by epigenetic mechanisms; nano-sized particles that might directly interact with subcellular components of the mitotic and meiotic machineries; cytostatic drugs in healthcare occupations; anticancer therapies potentially affecting the genetic integrity of gametes; continuously increasing electromagnetic field exposures with some sparse evidence of aneugenic activity; endocrine disruptors and their seemingly elusive effects in mouse oocytes, including the first evidence that prenatal exposure could affect meiotic nondisjunction in adult life. Hazards are considered for both somatic cells at risk of neoplastic transformation or tumour progression by chromosome loss and gain and germ cells at risk of heritable aneuploidies associated with spontaneous abortions or genetic diseases. Finally, possible synergistic interactions between environmental exposure and ageing or genetic predisposition are considered that could influence ultimate risks.

In vivo repair of DNA damage induced by X-rays in the early stages of mouse fertilization, and the influence of maternal PARP1 ablation.

The early pronucleus stage of the mouse zygote has been characterised in vitro as radiosensitive, due to a high rate of induction of chromosome-type chromosome abnormalities (CA). We have investigated the repair of irradiation induced double strand DNA breaks in vivo by γH2AX foci and first cleavage metaphase analysis. Breaks were induced in sperm and in the early zygote stages comprising sperm chromatin remodelling and early pronucleus expansion. Moreover, the role of PARP1 in the formation and repair of spontaneous and radiation-induced double strand breaks in the zygote was evaluated by comparing observations in C57BL/6J and PARP1 genetically ablated females. The results confirmed in vivo that the rate of chromosome aberration induction by X-rays was approximately 3-fold higher in the zygote than in mouse lymphocytes. This finding was related to a diminished efficiency of double strand break signalling, as shown by a lower rate of γH2AX radiation-induced foci compared to that measured in most other somatic cell types. The spontaneous frequency of CA in PARP1 depleted zygotes was slightly but significantly higher than in wild type zygotes. Also, these zygotes showed some impairment of the radiation-induced DNA Damage Response when exposed closer to the start of S-phase, revealed by a higher number of γH2AX foci and a longer cell cycle delay. The rate of chromosome aberrations, however, was not elevated over that of wild type zygotes, possibly thanks to backup repair pathways and/or selection mechanisms against damaged cells. When comparing with the literature data on irradiation induced CA in mouse zygotes in vitro, the levels of induction were strikingly similar as was the frequency of misrepair of double strand breaks (γH2AX foci). This result can be reassuring for in vitro human gamete and embryo handling, because it shows that culture conditions do not significantly affect double strand DNA break repair.

Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse.

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.

Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood.

Mice heterozygous for a p53 null mutation develop tumours induced by genotoxic carcinogens with a shorter latency than wild type mice and have been proposed as an alternate animal model for carcinogenicity testing. Some literature data suggest that p53+/- mice might also be more sensitive to the short-term effects of genotoxic agents and manifest a haploinsufficiency phenotype that could contribute to the higher tumour susceptibility. We have compared the induction of micronuclei in bone marrow and blood of p53+/- and p53+/+ isogenic mice after treatment with a single or multiple doses of melphalan (MLP), a crosslinking genotoxic carcinogen. We have also characterized the mechanism of micronucleus induction with CREST staining of kinetochore proteins to distinguish between chromosome break- and chromosome loss-induced micronuclei. Significant increases of micronucleated bone marrow polychromatic erythrocytes and blood reticulocytes were induced under all MLP exposure conditions. The frequency of micronucleated blood erythrocytes increased linearly with duration of exposure. Micronuclei were essentially a consequence of chromosome break events. After a single MLP dose, a significant reduction of the frequency of polychromatic erythrocytes in bone marrow of p53+/+ animals suggested the induction of cytotoxicity/cell cycle delay. This effect was not observed in p53+/- mice. We believe this finding to provide some evidence of a haploinsufficiency phenotype in the modulation of cell cycle/apoptotic pathways mediated by the p53 protein. In bone marrow of wild type mice, an increased effect of multiple MLP doses was detected over that of a single administration, whereas, in p53+/- mice, no differential effect was found of different exposure durations. Possibly, the probability of micronucleus formation increased under chronic exposure because of increased cell division in response to peripheral anemia and a reduction of p53 protein level had a small effect on cell cycle modulation and on such indirect mechanism of micronucleus induction. However, pairwise comparisons between the frequencies of cells with micronuclei in wild type and p53+/- mice under all exposure conditions did not show statistically significant differences, suggesting that the observed effects of p53 haploinsufficiency were weak and temporary and a higher/faster induction of irreversible chromosome damage could not account for the increased susceptibility of p53+/- mice to MLP-induced tumours.

Nondisjunction rates of mouse chromosomes involved in heterozygous Rb rearrangements measured by chromosome painting of spermatocytes. II. The effects of trivalent combinations and genetic background.

Chromosome specific nondisjunction rates were quantified by dual-colour FISH in spermatocytes II of Robertsonian heterozygous mice with different trivalent combinations or, alternatively, with different genetic backgrounds. We found that such factors do not influence the proneness to nondisjunction of specific chromosomes.

Cosegregation of Robertsonian metacentric chromosomes in the first meiotic division of multiple heterozygous male mice as revealed by FISH analysis of spermatocyte II metaphases.

Contrasting results (random segregation or cosegregation of isomorphic chromosomes) have been reported up to now on the segregation pattern of Robertsonian metacentric chromosomes of Mus musculus domesticus in multiple heterozygotes, using different approaches (karyotypical analysis of the progeny or of second meiotic metaphases). In the present contribution data are presented based on FISH (Fluorescence In Situ Hybridisation) analysis with telomeric probes, which allowed us to distinguish metacentric chromosomes from pairs of acrocentric chromosomes with their centromeric regions close to each other. Probes were hybridized to DAPI stained metaphases of spermatocytes II of mice heterozygous for two, three or four Robertsonian metacentrics in an all-acrocentric background, the karyotype of which has been reconstructed starting from laboratory strains. Isomorphic chromosomes tend to cosegregate (metacentrics with metacentrics, acrocentrics with acrocentrics); the values found for cosegregation have a clear even if moderate effect on the reproductive isolation caused by underdominant chromosomal rearrangements.

Nondisjunction rates of mouse specific chromosomes involved in heterozygous Rb rearrangements measured by chromosome painting of spermatocytes II. I. The effects of the number of trivalents.

Dual-colour FISH painting with alternative fluorescent chromosome-specific probes allowed us to distinguish chromosomes 1, 4, 6 and 14. The purpose was to check whether nondisjunction rates of specific chromosomes involved in heterozygous Robertsonian fusions are independent of the number of trivalents, or an epistatic effect among Rb chromosomes takes place affecting nondisjunction rates. Probes were used on DAPI-stained metaphases of spermatocytes II of laboratory strains of mice with reconstructed karyotypes heterozygous for one, two, three or four Robertsonian metacentrics in an all-acrocentric background. The existence of such epistatic interactions was not verified.

Induction and transmission of chromosome aberrations in mouse oocytes after treatment with butadiene diepoxide.

A study was conducted on the genotoxicity of butadiene diepoxide (DEB) in mouse oocytes. Superovulated female mice were injected intraperitoneally with DEB and mated with untreated males. Oocyte exposure occurred approximately 1.5 days before ovulation. DEB doses ranged between 26 and 52 mg/kg. Chromosome aberrations were scored in C-banded metaphases of one-cell embryos. The percentage of mated females, the average number of zygotes harvested per female, the frequencies of unfertilized oocytes and developmentally delayed zygotes did not reveal any overt sign of chemical toxicity which hindered the propensity of animals to mate or affected the ovulation, fertilization, or cell cycle progression of treated oocytes. A dose-dependent induction of chromosome aberrations was observed which was best fitted by a linear-quadratic equation. Half of all the aberrations transmitted by DEB-treated oocytes were chromatid-type breaks or exchanges. Among chromosome-type aberrations, double fragments for exceeded chromosome exchanges. This spectrum of structural aberrations differed markedly from what was previously observed in one-cell embryos conceived by DEB-treated sperm, where 97% were chromosome-type aberrations and 40% were dicentrics or translocations. This difference suggests that chromosome damage in one-cell embryos can be fixed by different mutagenic pathways influenced by the targeted gamete and its specific chromatin configuration. After exposure to the same dose, oocytes transmitted to one-cell embryos between 4 and 8 times fewer aberrations than DEB-treated sperm. While the rate of aberration induction suggests that female germ cells may be less at risk than mature sperm, especially at low-dose levels, the higher threshold for reproductive toxicity observed in female than in male mice may justify inclusion of data on female germ cell mutagenicity in the genetic risk assessment of butadiene exposure.

Nitrilotriacetic acid (NTA) induces aneuploidy in Drosophila and mouse germ-line cells.

The ability of nitrilotriacetic acid (NTA) to induce aneuploidy was studied in the germ line of both Drosophila and the mouse. The Free Inverted X Chromosomes (FIX) genetic system, adopting a brooding scheme, was used to detect induced aneuploidy in Drosophila, and a cytogenetic method based on chromosomal counting in secondary spermatocytes was used in the mouse. In Drosophila a highly significant (P less than 0.001) increase of aneuploidies was produced by NTA (5 x 10(-2) M), which was greater than that produced by colchicine (7.5 x 10(-6) M) and 5-fluorodeoxyuridine (10(-4) M), which were used as positive controls. Brooding effects were observed with NTA, which produced a maximum induction of chromosomal gain in brood I, suggesting a possible stage-specific action during meiosis. The ability of NTA (275 mg/kg body weight) to induce meiotic aneuploidy (hyperhaploidy) also was confirmed in the mouse (P less than 0.001), where all the aneuploidies detected were attributable to treatment of the metaphase I stage.

Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes.

Young superovulated female mice were injected i.p. with single doses of vinblastine sulfate just before the onset of the first meiotic division. Secondary oocytes, fixed one by one on a slide, were cytogenetically scored. Evidence of the meiotic arresting activity of vinblastine was produced by the observation of increasing frequencies of M1-arrested oocytes and by the presence of undegenerated chromosome sets of first polar bodies. When the first meiotic division could be undertaken chromosome malsegregation occurred with high frequency, both in terms of aneuploidy and polyploidy. M1-blocked and polyploid oocytes have been interpreted as the consequence of irreversible damage to the spindle induced by vinblastine through its binding on tubulin low-affinity sites; this reaction, in fact, causes microtubule crystallization. According to this mechanism, dose-effect relationships of both phenomena show a threshold at 0.45 mg/kg. On the other hand, the incidence of aneuploid oocytes is correlated with meiotic delay, as detected by the delayed degeneration of polar bodies, and increases linearly with dose. Both phenomena are, therefore, stochastic and can be referred to the binding of the chemical on tubulin high-affinity sites, which is known to cause tubulin depolymerization in a colchicine-like way.

Cytogenetic investigation of chemically-induced aneuploidy in mouse spermatocytes.

This paper discusses a test system in which mouse spermatocytes are analyzed for aneuploidy induction after mice are treated with various agents. Included in this report are methods and procedures of the assay, criteria for determination of aneuploidy induction, considerations for dose-response and stage-specific actions of agents that cause aneuploidy, and finally, advantages and disadvantages of this test system.

Meiotic non-disjunction induced by fission neutrons relative to X-rays observed in mouse secondary spermatocytes. II. Dose-effect relationships after treatment of pachytene cells.

(C57B1/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons (from 0.11 to 0.72 Gy) or 250 kV X-rays (from 0.25 to 3 Gy) and sacrificed 5 days later. Chromosome preparations of secondary spermatocytes, irradiated at the stage of pachytene, were analysed and the incidence of hyper-haploidies and chromosome fragments was recorded. Data on numerical aberrations were fitted by highly significant linear relationships for both types of radiation. A relative biological effectiveness (RBE) value of 5.65 was estimated by the ratio between the slopes of the two regression lines. The same linear fitting was applied to frequencies of cells with fragments, even if in this case other types of functions could not be excluded. An RBE value was estimated in the same way as for numerical aberrations and yielded a comparable figure of 5.23. A significant correlation was also found between the incidence of numerical and structural aberrations, which points to the chromosome itself as the prevalent target for radiation-induced non-disjunction (ND). In addition, the highly significant linearity of the dose-effect relationship observed for the induction of aneuploidies suggests, as the simplest hypothesis, a single-hit mechanism of radiation action, possibly through pre-non-disjunctional damage to the centromeric region, rather than an indirect induction of segregational difficulties after primarily induced chromatid interchanges.

Vinblastine-induced numerical chromosome changes and selection processes in mouse bone marrow cells.

The effect of selection processes operating on chemically induced aneuploid and polyploid cells was studied in mouse bone marrow cells at their third generation after a single i.p. treatment with vinblastine (VBL). Bromodeoxyuridine (BrdUrd)-labeled metaphases were analyzed for chromosome number and the frequencies of aneuploid and polyploid cells recorded at 2 different times, both within the third cell cycle after VBL treatment. Cell-cycle progression was analyzed for both control and treated mice at the 2 fixation times. Our data suggest that polyploid cells and possibly also cells with numerous additional chromosomes could have a cell cycle longer than that of diploid cells and cells hyperploid for 1-2 additional chromosomes. Both hyperploid and polyploid cells seem to have a reduced probability of undergoing further mitoses, as shown by the reduction of their frequencies at the third cell cycle, when compared to the frequencies observed in the second cell cycle after the same VBL treatment.

Genotoxic effects of butadiene in mouse lung cells detected by an ex vivo micronucleus test.

Lung fibroblasts from BD-exposed mice have been analysed for the occurrence of micronuclei. Primary cultures set up 24h after the end of exposure were treated with cytochalasin B and micronuclei scored in binucleate cells. A three-fold statistically significant increase of micronucleated cells was detected after exposure to 500ppm, the lowest tested concentration. A linear dose effect relationship was observed between 500 and 1300ppm. Immunofluorescent staining of kinetochore proteins was applied to distinguish between acentric micronuclei produced by chromosome breaks and micronuclei containing a centromeric region, most likely induced by chromosome loss. A statistically significant increase of both types of MN in 1300ppm-exposed females and a significant increase in centromeric MN in 500ppm-exposed males were detected. These data demonstrate that an intermediate of BD metabolism with a potential for clastogenic and aneugenic effects is active in lung cells after inhalation exposure. These effects can play a role in the initiation and promotion of BD-induced lung tumours.

Diepoxybutane cytotoxicity on mouse germ cells is enhanced by in vivo glutathione depletion: a flow cytometric approach.

Diepoxybutane is one of the key metabolites of butadiene, a compound of high environmental and occupational concern. The effects of diepoxybutane on mouse reproductive cells have been previously characterized by flow cytometry demonstrating a specific, dose-dependent cytotoxicity for differentiating spermatogonia. It is known that butadiene epoxides, deriving from butadiene bioactivation by cytochrome P450-monooxygenase systems, can be enzymatically conjugated to glutathione by glutathione S-transferases. In this paper, we tested the hypothesis whether a pretreatment with phorone, a well-known intracellular glutathione depleter, would enhance the germ cell cytotoxicity of diepoxybutane. Results were consistent with an active role played in vivo by the glutathione-detoxifying system, as diepoxybutane cytotoxicity was increased after chemically induced reduction of glutathione concentration.

In vivo studies on chemically induced aneuploidy in mouse somatic and germinal cells.

Within the context of a coordinated program to study aneuploidy induction sponsored by the European Community, nine chemicals were tested in mouse bone marrow and spermatocytes after intraperitoneal injection. In somatic cells, cell progression delay, hyperploidy, polyploidy induction and induction of micronucleated polychromatic erythrocyte (MnPCE) were studied. In germ cells hyperploidy induction was evaluated. The chemicals selected were: colchicine (COL), econazole (EZ), hydroquinone (HQ), thiabendazole (TB), diazepam (DZ), chloral hydrate (CH), cadmium chloride (CD), pyrimethamine (PY) and thimerosal (TM). Using literature data on c-mitotic effects in bone marrow as a reference, the same doses were tested in somatic and germ cells in order to compare the effects induced. Bone marrow cells were sampled 18 or 24 h after treatment. Germ cells were sampled 6, 8 or 18 h after treatment. Effects of COL and HQ in bone marrow have been reported elsewhere. Somatic effects were induced by CH (hyperploidy and cell cycle lengthening), TB (MnPCEs and cell cycle lengthening) and by PY (MnPCEs). EZ, DZ, CD and TM did not induce any kind of somatic effects. An increase in the incidence of hyperploid spermatocytes was induced by COL, at three dose levels, and by one dose of HQ and TB. All the other chemicals did not induce germinal aneuploidy at any dose or time tested. The hyperploidy control frequency ranged between 0.4 and 1.0% in somatic cells and from 0.3 to 0.9% in germ cells. In both somatic and germ cells, the maximum yield of induced hyperploidy did not exceed 3.5%. The time period of target cell sensitivity is probably restricted and this, associated with the heterogeneity and the asynchrony of cellular maturation processes, may account for our data. Under these circumstances, the negative data should be interpreted with some caution, particularly in germ cells, where additional indicators of chemical-cell interaction and cell cycle effects were not provided by standardized approaches. The possibility of increasing the size of analyzed cell samples could be considered in the light of automatic scoring procedures.

Origin of aneuploidy in relation to disturbances of cell-cycle progression. I. Effects of vinblastine on mouse bone marrow cells.

Vinblastine (VBL) was tested in the mouse for induction of chromosome malsegregation in bone marrow cells. The occurrence of aneuploidy and polyploidy was correlated with cell-cycle kinetics measured by DNA labelling with bromodeoxyuridine (BrdUrd). Sister-chromatid exchanges (SCE) were also detected. A dose-dependent lengthening of the cell cycle was induced in the dose range of 0.9-4.5 mg/kg body weight, up to a complete inhibition of cell-cycle progression (100% of metaphases were arrested before completion of the first mitotic division following a recovery time of 18 h, compared with 8% in the controls). Both aneuploidy and polyploidy were induced. Aneuploid metaphases were grouped into 2 classes, those with no more than 2 extra chromosomes and those with 3-10 extra chromosomes. The frequencies of cells with severe aneuploidy and polyploidy increased considerably when second-generation cells were sampled at a recovery time of 24 h. This observation suggested that gross chromosome imbalances occur preferentially after a period of mitotic arrest, probably as a consequence of multipolar spindles or failure of proper spindle assembly. Non-disjunction of single chromosomes arises independently of the mitotic block. A slight increase in SCE frequency was observed only at a recovery time of 18 h. This study may provide information on the kinetics and mechanisms of origin of VBL-induced numerical aberrations in vivo.

Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. I. Cytogenetic analysis of metaphase II oocytes.

Griseofulvin (GF) was tested in female mouse germ cells for the induction of aneuploidy and meiotic arrest. Superovulated mice were orally treated with 200, 666, 1332 or 2000 mg/kg in olive oil at the time of human chorionic gonadotrophin (HCG) injection and were sacrificed 18 h later. A dose-dependent increase in the frequency of metaphase I (M I) arrested oocytes was observed (maximum of 70%). Aneuploidy was not significantly induced. Also, the kinetics of meiotic progression up to the metaphase II (M II) stage was studied in untreated mice in order to correlate the time of treatment with the time of the first meiotic division. The results demonstrate that the majority of cells was treated with GF approximately 8 h before the M I stage. A second series of experiments were performed to test GF effects at a different treatment time. Doses of 200, 666 or 2000 mg/kg were administered 2 h post HCG. As in the first series of experiments, the animals were sacrificed 18 h post HCG. The results, compared with those obtained in the first experimental series, showed an inverse trend for meiotic arrest and aneuploidy induction. The frequency of M I arrested oocytes dropped from a maximum of 70% to a maximum of 20%, while, at the latest treatment time, a dose-dependent increase in the frequency of hyperploid oocytes was observed up to 56% aberrant cells at 2000 mg/kg. Altogether the results suggest that the arrest of meiotic division and the induction of aneuploidy by GF are caused by interaction with different targets or different developmental stages of the same target. In conclusion, GF has been shown to induce aneuploidy during the first meiotic division in a dose-related manner, together with other effects such as polyploidy, developmental delay and meiotic arrest. Also, these findings demonstrate that the sensitivity of the oocyte target(s) may be restricted to a specific time period and that a correct experimental protocol is critical for assessing the aneugenic activity of a chemical.

Griseofulvin-induced aneuploidy and meiotic delay in female mouse germ cells. II. Cytogenetic analysis of one-cell zygotes.

The effects of griseofulvin (GF) upon the first meiotic division of female mouse germ cells were evaluated by cytogenetic analysis of first-cleavage (1-Cl) zygotes. The present study is an extension of an investigation that began with the cytogenetic analysis of metaphase II (M II) oocytes. Different doses (200, 666, 1332, 2000 mg/kg) were tested by oral administration of GF to superovulated animals either at the time of human chorionic gonadotrophin (HCG) injection or 2 h post HCG. When GF was given at the time of HCG, significant dose-dependent increases of different types of cytogenetically abnormal cells were found. These included zygotes containing ostensibly female-derived M I or M II arrested chromosomes and polyploid zygotes. The total yields of these aberrations were 2.9, 4.3, 26.2, 60.6, and 64.1% for control, 200, 666, 1332, and 2000 mg/kg, respectively. The origin of these zygotes was attributed to the fertilization of oocytes that had been previously arrested at M I. No significant induction of hyperploidy was detected. Developmentally abnormal zygotes were still observed when GF was administered 2 h post HCG, although their frequencies were significantly lower than in the first series of experiments. The yields of developmentally abnormal zygotes were 49, 10.2, and 23.6% at 200, 666, and 2000 mg/kg. Additionally, a dose-dependent increase in the frequency of hyperploid zygotes was detected up to a maximum of 36.5% at 2000 mg/kg. These results confirm the cytogenetic observations from M II oocytes after GF treatment under the same experimental conditions; namely, a dramatic change in the oocyte target susceptibility to GF occurred within a short time period. Also, the present study demonstrated that most of GF-induced aneuploid oocytes were fertilized and reached first-cleavage metaphase.

DNA adducts in mouse testis and lung after inhalation exposure to 1,3-butadiene.

1,3-Butadiene is a widely used industrial chemical and also an environmental contaminant. Recent findings have shown that butadiene can also be a male germ cell mutagen. In this study, DNA adduct formation in testis and lung has been explored by using N7-alkylated guanines as a marker of biological effective dose. The adducts measured were the four structurally different guanine N7-adducts alkylated by butadienemonoepoxide, the main metabolite of 1,3-butadiene. This study demonstrates the dose-dependent adduct formation in lung and testis. At lower exposures (50 and 200 ppm) the adduct levels were about the same in the two organs, but at 500 ppm the adduct level was significantly (p < 0.03) higher in testis than in lung. The enantiomeric composition of the adducts detected was also different. In lung, all 4 possible adducts were present (S-C-1" dominating, 49%), but in testis only two out of four adducts were detected (S-C-2" being the most abundant adduct, 71%). These novel observations indicate that the DNA repair is different in these two organs studied and that heritable genetic effects observed may be mediated through the DNA adducts.