Toxicity and genotoxicity induced by abacavir antiretroviral medication alone or in combination with zidovudine and/or lamivudine in Drosophila melanogaster.

Abacavir (ABC), zidovudine (AZT), and lamivudine (3TC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) widely used as combination-based antiretroviral therapy against human immunodeficiency virus. Despite effective viral suppression using NRTI combinations, genotoxic potential of NRTIs can be increased when administered in combination. This study investigated the toxic and genotoxic potential of ABC when administered alone or in combination with AZT and/or 3TC using the somatic mutation and recombination test in Drosophila melanogaster. This test simultaneously evaluated two events related to carcinogenic potential: mutation and somatic recombination. The results indicated that ABC was responsible for toxicity when administered alone or in combination with AZT and/or 3TC. In addition, all treatment combinations increased frequencies of mutation and somatic recombination. The combination of AZT/3TC showed the lowest genotoxic activity compared to all combinations with ABC. Therefore, our results indicated that ABC was responsible for a significant portion of genotoxic activity of these combinations. Somatic recombination was the main genetic event observed, ranging from 83.7% to 97.7%.

No association between body mass index and sperm DNA integrity.

STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including mainly subfertile men, have shown conflicting results regarding the influence of overweight and obesity on sperm DNA integrity. STUDY DESIGN, SIZE, DURATION: This cross-sectional study was based on semen samples from 1503 men from the general population. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included two cohorts (cohort A and B) of military recruits (n = 275, n = 304, respectively), one group (cohort C) of fertile men and men without known fertility problems (n = 724), and one group (cohort D) of men between 19 and 40 years without known fertility problems (n = 200). In all cohorts, data were available on BMI, DFI as measured by the sperm chromatin structure assay (SCSA), standard semen characteristics, and potential confounders (age, abstinence time, smoking habits). The subjects were categorized according to BMI into four groups: underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)), overweight (25.0-29.9 kg/m(2)) and obese (≥30.0 kg/m(2)). Using a linear regression model, the inter-group differences in DFI were calculated. Furthermore with the normal-weight group as the reference, the odds ratios (ORs) for DFI > 20% and DFI > 30%, were calculated for the other groups. Calculations were made for the material as a whole and after exclusion of cohort C which included proven fertile men. MAIN RESULTS AND THE ROLE OF CHANCE: We found that normal-weight men had significantly higher DFI than overweight men, with a mean difference of 1.13% (95% CI: 1.05-1.22%); P = 0.001). Overweight men had a reduced risk of having DFI ≥ 20% and DFI ≥ 30%, compared with normal-weight men; adjusted odds ratio (OR) = 0.61 (95% CI: 0.42-0.88; P < 0.01) and adjusted OR = 0.48 (95% CI: 0.28-0.84; P < 0.01), respectively. When excluding cohort C, the statistical significance was lost. Regarding standard semen parameters, we found that obese men had a higher percentage of progressive motile spermatozoa than normal-weight men; mean difference 1.15% (95% CI: 1.02-1.30%, P < 0.05) but the significance was lost when excluding cohort C. All other standard semen parameters were unaffected by BMI. LIMITATIONS, REASONS FOR CAUTION: A main limitation might be the cross-sectional nature of the data. Furthermore our study included a significant proportion of men with proven fertility (75% of cohort C, n = 550), and could therefore be biased toward fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our study indicates that overweight per se is not associated with a higher level of sperm DNA damage. STUDY FUNDING/COMPETING INTERESTS: This research has been given grants from the following: EU 5th and 7th framework program (Inuendo and Clear projects, [Contracts no. QLK4-CT-2001-00202 and FP7-ENV-2008-1-226217)]), the Swedish Research Council (Grants No. 2007-2590, 521-2004-6072 and 521-2002-3907); the Swedish Governmental Funding for Clinical Research, Skåne county council's research and development foundation, MAS Funds, University Hospital MAS Foundation in Malmö, Crafoordska Fund, Ove Tulefjords Fund, Foundation for Urological Research, Fundacion Federico SA, and Gunnar Nilssons Cancer Fund. The authors declare that there are no conflicts of interest.

Testicular cancer and sperm DNA damage: short- and long-term effects of antineoplastic treatment.

The aim of this study was to investigate sperm DNA damage induced by chemo- and radiotherapy in patients with testicular cancer to provide data on the extent and persistence of nuclear damage that might affect individual reproductive potential. We evaluated pre- and post-antineoplastic treatment sperm DNA integrity, expressed as DNA Fragmentation Index (DFI), in a large caseload of testicular cancer patients by sperm chromatin structure assay. The mean total DFI for all patients at T0 was 18.0 ± 12.5%. Sperm chromatin profile was markedly impaired at T3 (27.7 ± 17.4%) and T6 (23.2 ± 15.3%), improving considerably at T12 and T24 (14.0 ± 8.9% and 14.4 ± 10.3%). After chemotherapy, we found a marked increase in DFI at T3 and T6 and a significant reduction at T12 and T24 in comparison with the baseline. In contrast, DFI increased at T3 and T6 after radiotherapy but the subsequent reduction was far less marked, reaching baseline values at T12 and T24. Finally, post-treatment DNA damage was not age or histotype dependent, but was more marked in the advanced stage of cancer. In this study, we showed that the chromatin profile may be affected in the months immediately following the end of the treatment, improving after 12-24 months. Our results thus indicate that post-treatment DNA damage is influenced both by the type and intensity of the therapy and by the pathological and clinical stage of the disease.

Non-linear association between androgen receptor CAG and GGN repeat lengths and reproductive parameters in fertile European and Inuit men.

Recently the dogma that there is an inverse linear association between androgen receptor (AR) CAG and GGN polymorphisms and receptor activity has been challenged. We analysed the pattern of association between 21 male reproductive phenotypes and AR CAG/GGN repeat lengths in 557 proven-fertile men. A linear association was only found between sperm DNA fragmentation index (DFI) and CAG length, and between inhibin B and GGN length. Men with longer CAG then the reference (22-24), had higher oestradiol levels, whereas men with shorter CAG stretches had a higher DFI and a higher proportion of Fas-positive germ cells. Subjects with either short or long CAG had increased seminal levels of prostate-specific antigen and neutral α-glucosidase activity. Compared to men with the median GGN length of 23, those with shorter GGN repeats had higher levels of inhibin B, higher proportions of normal and progressive sperm, and a higher fraction of Fas-positive sperm, while men with longer GGN had higher oestradiol levels. These data indicate that at least for some markers of male reproductive function the association with CAG or GGN repeat length is curvilinear.

Exposures that may affect sperm DNA integrity: two decades of follow-up in a pregnancy cohort.

Prenatal lifestyle exposures are linked to alterations in conventional semen characteristics. Sperm DNA integrity is another marker of semen quality shown to be altered in mice prenatally exposed to chemicals. From a Danish pregnancy cohort established in 1984-1987, sons were selected for a follow-up study in 2005-2006. We examined associations between prenatal and current lifestyle exposures and DNA fragmentation index (DFI) among 337 men. Sons of overweight mothers had 22% (95% CI: -3; 52) higher DFI than sons of normal weight mothers and sons of parents with a TTP >12 months had 14% (95% CI: -4; 34) higher DFI than sons of parents with a TTP of 0-6 months. Abstinence time was positively associated with DFI (p<0.005). Overweight men had higher DFI compared to normal weight men, however, statistically insignificantly. In conclusion, results indicate that DFI is affected by prenatal exposures, but confidence limits are wide and results statistically insignificant.

Sperm DNA integrity in cancer patients: the effect of disease and treatment.

As oncological treatment might impair the patients' fertility, male cancer patients are offered to cryopreserve semen prior to treatment. Impaired sperm DNA quality is associated with reduced fertility, and in case of assisted reproduction, sperm DNA integrity may have an impact on choice of method. Therefore, we have assessed sperm DNA integrity in cancer patients, comparing pre- and post-treatment quality. Sperm DNA integrity was investigated in cryopreserved semen from 121 cancer patients, the predominating diagnoses were germ cell cancer (GCC) and Hodgkin's lymphoma (HL). Post-treatment samples, with a median follow-up of 3 years, were analysed for 58 of the men, allowing a pre- and post-treatment analysis on an individual basis. Sperm DNA integrity was assessed using the Sperm Chromatin Structure Assay and expressed here as the DNA Fragmentation Index (DFI%). One hundred and thirty-seven fertile men served as controls. Before treatment, GCC (n = 84) and HL (n = 18) patients had higher DFI% than controls (n = 143) with a mean difference of 7.7 (95% CI 3.2-8.8) and 7.0 (95% CI 2-12), respectively. The same trend was observed for other cancer diagnoses, but without reaching statistical significance (mean difference 3.6, 95% CI -1.2 to 8.4). No increase was seen in DFI% comparing pre- and post-treatment semen, regardless of treatment modality. A moderate elevation of DFI% was observed in cryopreserved semen from cancer patients. Oncological treatment, generally, did not induce any increase in DFI. These findings should be considered when discussing the utilization of pre-treatment cryopreserved semen vs. post-treatment fresh sperm in cancer patients undergoing assisted reproduction.

Inhibitory effects of water extract of propolis on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster.

Propolis is a substance produced by honeybees (Apis mellifera L.). Its components are strong antioxidants and free radical scavengers. The aim of this study was to evaluate the protective effects of a water extract of Brazilian green propolis (WEP) combined with the antitumor agent doxorubicin (DXR) on Drosophila melanogaster wing cells through the somatic mutation and recombination test (SMART). Two different crosses were used: The standard (ST) cross and the high bioactivation (HB) cross. The HB cross is characterized by a constitutively enhanced level of cytochrome P450 which leads to an increased sensitivity to a number of promutagens and procarcinogens. Larvae obtained from these two crosses were chronically treated with different concentrations of WEP (12.5,25.0 and 50.0 mg/mL) alone or combined with DXR (0.125 mg/mL). The results obtained with the two different crosses were rather similar. Neither toxicity nor genotoxicity were observed in WEP treated series. Simultaneous treatment with different concentrations of WEP and DXR led to a reduction in the frequency of recombination compared to the treatment with DXR alone. This anti-recombinogenic effect was proportional to the concentrations applied, indicating a dose-response correlation and can be attributed to the powerful scavenger ability of WEP.

Prenatal cannabis exposure increases heroin seeking with allostatic changes in limbic enkephalin systems in adulthood.

BACKGROUND: Prenatal cannabis exposure is a growing concern with little known about the long-term consequences on behavior and neural systems relevant for reward and emotional processing. METHODS: We used an animal model to study the effects of prenatal exposure to Delta(9)-tetrahydrocannabinol (THC) on heroin self-administration behavior and opioid neural systems in adult males (postnatal day 62). Rats were exposed to THC (.15 mg/kg) or vehicle from gestational day 5 to postnatal day 2. RESULTS: Both pretreatment groups showed similar heroin intake, but THC-exposed rats exhibited shorter latency to the first active lever press, responded more for low heroin doses, and had higher heroin-seeking during mild stress and drug extinction. THC exposure reduced preproenkephalin (PENK) mRNA expression in the nucleus accumbens during early development, but was elevated in adulthood; no adult striatal changes on preprodynorphin mRNA or PENK in caudate-putamen. PENK mRNA was also increased in the central and medial amygdala in adult THC-exposed animals. THC animals had reduced heroin-induced locomotor activity and nucleus accumbens mu opioid receptor coupling. CONCLUSIONS: This study demonstrates enduring effects of prenatal THC exposure into adulthood that is evident on heroin-seeking behavior during extinction and allostatic changes in mesocorticolimbic PENK systems relevant to drug motivation/reward and stress response.

Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p'-DDE in European and Inuit populations.

Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to be investigated in order to fully assess the population variations observed.

Sperm DNA integrity in testicular cancer patients.

BACKGROUND: We evaluated the impact of testicular germ cell cancer (TGCC), its treatment and length of follow-up on sperm DNA integrity. METHODS: In 96 TGCC patients, semen was collected at specific intervals until 5 years after treatment. Sperm DNA integrity was assessed by the sperm chromatin structure assay (SCSA, n = 193) and by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL, n = 159) assay. Results were expressed as DNA fragmentation index (DFI). Controls comprised of 278 military conscripts. RESULTS: Post-surgery testicular cancer (TC) patients did not differ from controls. Compared with pretreatment values, radiotherapy induced a transient increase in SCSA(DFI) (medians: 12 versus 19%; P = 0.03), normalizing after 3-5 years. One year or more after therapy, 5/13 (38%) of normozoospermic, irradiated patients had SCSA(DFI) >27% compared with 7% of normozoospermic controls (P = 0.002). More than two cycles of chemotherapy decreased DFI 3-5 years post-therapy (median SCSA(DFI): 12 versus 9.1%, P = 0.02; median TUNEL(DFI): 11 versus 7.5%, P = 0.03). CONCLUSION: Irradiation increases sperm DNA damage 1-2 years after treatment, and 38% of irradiated patients with normozoospermia had high (>27%) DNA damage, which may affect the sperm-fertilizing ability. TC per se is not associated with an increase of DFI, and DFI is reduced by three or more cycles of chemotherapy.

Cannabinoid mechanism in reinstatement of heroin-seeking after a long period of abstinence in rats.

Because opioid and cannabinoid systems have been reported to interact in the modulation of addictive behaviour, this study was aimed at investigating the ability of cannabinoid agents to reinstate or prevent heroin-seeking behaviour after a prolonged period of extinction. In rats previously trained to self-administer heroin intravenously, non-contingent non-reinforced priming administrations of heroin and cannabinoids were presented after long-term extinction, and lever pressing following injections was observed. Results showed that: (i) intravenous priming infusions of heroin (0.1 and 0.2 mg/kg) lead to reinstatement of drug-seeking behaviour; (ii) intraperitoneal priming injections of the central cannabinoid receptor agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazinyl) (1-naphthalenyl)methanonemesylate (WIN 55,212-2, 0.15 and 0.3 mg/kg) and (-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940, 0.05 and 0.1 mg/kg), but not delta9-tetrahydrocannabinol (delta9-THC, 0.1-1.0 mg/kg), effectively restored heroin-seeking behaviour; (iii) intraperitoneal priming injection of the central cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 0.3 mg/kg) did not reinstate responding, but (iv) completely prevented heroin-induced reinstatement of drug-seeking behaviour. Moreover, heroin-seeking behaviour was still present for a few days following cannabinoid primings, indicating a long-lasting effect of cannabinoids on responding for heroin. These findings indicate that relapse to heroin after an extended drug-free period is triggered by cannabinoid agonists and that SR 141716A prevents drug-seeking behaviour, suggesting that the use of the cannabinoid antagonist could have some therapeutic benefits in heroin-induced relapse.

The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA).

BACKGROUND: The sperm chromatin structure assay (SCSA) provides an objective assessment of sperm chromatin integrity, which is essential for normal sperm function. SCSA is valuable as a fertility marker in epidemiological studies and in the clinical situation. Little is known about the impact of testicular and post-testicular function on SCSA parameters. METHODS: Ejaculates from 278 military conscripts of median age 18.1 (range 18-21) years were included. Levels of reproductive hormones, the length of the CAG repeat of the androgen receptor gene, sperm concentration, abstinence period and biochemical parameters of epididymal and accessory sex gland secretions were correlated to the SCSA parameters, DNA fragmentation index (DFI) and highly DNA stainable (HDS) cells. RESULTS: Negative correlations were found between sperm concentration and DFI (r = -0.119, P = 0.049) and HDS (r = -0.513, P < 0.0001). DFI was negatively correlated with levels of estradiol (r = -0.19, P = 0.002) and free testosterone (r = -0.13, P = 0.03). DFI also correlated positively with abstinence time (r = 0.17, P = 0.005), and with seminal concentrations of fructose (r = 0.18, P = 0.004) and zinc (r = 0.12, P = 0.04). CONCLUSIONS: Sex steroid production, spermatogenic function, abstinence time and seminal vesicle function appear to impact on sperm chromatin integrity and thereby on sperm fertilizing capacity. These findings may improve present understanding of the pathophysiology of male infertility.

Flow cytometric DNA ploidy, p53, PCNA, and c-erbB-2 protein expressions as predictors of survival in surgically resected gastric cancer patients.

In order to determine retrospectively the impact of some cytometric and immunohistochemical parameters on the overall survival of gastric cancer patients treated with surgery alone, paraffin-embedded tumor samples from 137 gastric carcinoma patients undergoing curative resection from 1987-1993 were analyzed by flow cytometry (FCM) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). FCM-derived parameters were DNA ploidy and fraction of S-phase cells (SPF). Multiple regression analysis was applied to determine the prognostic significance of the conventional clinicopathologic findings together with the flow cytometric and immunohistochemical parameters on overall survival. When all parameters were entered simultaneously into the Cox regression model, stage and DNA ploidy (DNA index >1.35) clearly emerged as the only independent prognostic factors. When the stages were analysed separately, the independent prognostic factors resulted DNA ploidy in early stages (I-II) and grading in stage IIIA tumors. For stage IIIB tumors, no independent prognostic factor was found. These results indicate that the DNA ploidy pattern is a valuable predictor of survival in curatively resected gastric cancer patients, especially when less advanced tumors are taken into consideration.

Change in semen quality and sperm chromatin structure following occupational styrene exposure. ASCLEPIOS.

OBJECTIVES: Organic solvents have been suspected to exert detrimental effects on human spermiogenesis. Styrene, which is both mutagenic and neurotoxic, was selected as a suitable organic solvent for further assessment of a possible effect on semen quality and sperm DNA damage. SUBJECTS AND METHODS: Semen samples were collected from 23 reinforced plastics workers at the time of employment and after 6 months of styrene exposure and from 21 nonexposed farmers. Intra-individual changes in conventional semen parameters and sperm-DNA denaturation patterns were related to the internal dose of styrene exposure as measured by postshift urinary mandelic acid. RESULTS: A statistically significant decline in sperm density was seen during styrene exposure from 63.5 to 46.0 million sperm/ml, whereas no decline was seen in the nonexposed subjects. The total sperm count was almost halved from an initial value of 175 million sperm/ejaculate. However, no relationship was apparent when the sperm parameters were related to internal levels of exposure. However, an exposure-response relationship was shown for DNA-denaturation patterns, but the numbers were small. CONCLUSION: A declining sperm count following styrene exposure is suggested. However, the findings of the internal and external comparisons are inconsistent, and this may be due to the high intraindividual variability of semen parameters and the limited study size but may also be attributable to a weak internal exposure gradient. Spermatogenesis may be vulnerable to styrene exposure. However, due to the small numbers these findings are only preliminary.

Different effects of omega-conotoxin on penile erection, yawning and paraventricular nitric oxide in male rats.

A dose of apomorphine or oxytocin that induces penile erection and yawning increases nitric oxide production in the paraventricular nucleus of the hypothalamus, as determined by the increase in NO2- and NO3- concentration induced by these substances in the paraventricular dialysate obtained from male rats. All the above responses were prevented by a dose of omega-conotoxin-GVIA as low as 5 ng. This potent inhibitor of N-type Ca2+ channels was injected into the paraventricular nucleus 15 min before apomorphine (50 ng) or oxytocin (10 ng). In contrast, omega-conotoxin was ineffective when the above responses were induced by N-methyl-D-aspartic acid (50 ng). The peptide toxin (5 ng) was also ineffective on the penile erection and yawning induced by the nitric oxide donors sodium nitroprusside (50 microg) or hydroxylamine (50 microg), injected into the paraventricular nucleus. The present results suggest that omega-conotoxin-sensitive Ca2+ channels are involved in the activation of nitric oxide synthase, penile erection and yawning induced by apomorphine and oxytocin, but not by N-methyl-D-aspartic acid, at the paraventricular level.

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios.

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.

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.

Reproductive toxicity of 1,3-butadiene in the mouse: cytogenetic analysis of chromosome aberrations in first-cleavage embryos and flow cytometric evaluation of spermatogonial cell killing.

Reproductive effects of 1,3 butadiene inhalation have been evaluated in male mice by reduction of post-meiotic germ cells, alteration of sperm chromatin structure and transmission of chromosome aberrations to one-cell embryos. Animals were exposed for 5 consecutive days for 6 h per day to butadiene concentrations of 130, 500 or 1300 ppm. The testicular fraction of post-meiotic germ cells was measured by flow cytometric analysis on the basis of their DNA content. Round spermatids were discriminated from mature, elongated spermatids by their different degree of chromatin condensation. Butadiene-induced cytotoxic effects on differentiating spermatogonia were shown by a concentration-dependent decrease of round spermatids occurring 21 days after chemical exposure, confirmed by a similar decrease of elongated spermatids measured in testes sampled 7 days later. Statistically significant effects were seen already at 130 ppm. An incomplete repopulation of the elongated spermatid compartment observed 35 days after exposure to 1300 ppm suggested that, at the highest concentration tested, butadiene toxicity extended to stem cells. Alterations of sperm chromatin were revealed by its increased sensitivity to acidic denaturation in situ. The percentage of abnormal sperm was significantly increased after butadiene exposure of differentiating spermatogonia and spermatocytes. This suggested the induction of persistent effects interfering with chromatin remodelling during spermiogenesis. Chromosome-type structural aberrations were significantly elevated in first-cleavage embryos conceived by males mated during the first and second week after the end of exposure. The lowest effective tested concentration was 500 ppm, the same reported for dominant lethal induction under identical exposure conditions. As in the dominant lethal assay, the effect of this dose was confined to exposed sperm, while both sperm and late spermatids were affected by the inhalation of 1300 ppm. A quantitative comparison between the effects induced by intraperitoneal injections of diepoxybutane or butadiene inhalations suggested that other reactive intermediates, in addition to diepoxybutane, might contribute to mediate butadiene-induced reproductive toxicity.