Subclinical markers of cardiovascular toxicity of benzene inhalation in mice.

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 µM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4+ and CD8+ T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene.

Toxicological responses of BEAS-2B cells to repeated exposures to benzene, toluene, m-xylene, and mesitylene using air-liquid interface method.

In order to reduce exposure to toxic chemicals, the European REACH regulation (1907/2006) recommends substituting toxic molecules with compounds that are less harmful to human health and the environment. Toluene is one of the most frequently used solvents in industries despite its toxicity. The objective of this study is to better understand and compare the toxicity of toluene and its homologues in a bronchial cell model. Thus, human bronchial BEAS-2B cells were exposed to steams of toluene, m-xylene, mesitylene (1,3,5-trimethylbenzene), and benzene (20 and 100 ppm). Exposure was carried out using an air-liquid interface (ALI) system (Vitrocell) during 1 h/day for 1, 3, or 5 days. Cytotoxicity, xenobiotic metabolism enzyme gene expression, and inflammatory response were evaluated following cell exposures. BEAS-2B cell exposure to toluene and its homologues revealed the involvement of major (CYP2E1) and minor metabolic pathways (CYP1A1). A late induction of genes (EPHX1, DHDH, ALDH2, and ALDH3B1) was measured from Day 3 and can be linked to the formation of metabolites. An increase in the secretion level of inflammatory markers (TNF-α, IL-6, IL-8, MCP-1, and GM-CSF) was also observed. In parallel, regulation between inflammatory mediators and the expression of transmembrane glycoprotein mucin MUC1 was also studied. This in vitro approach with ALI system points out the relevance of conducting repeated exposures to detect potential late effects. The difference recorded after cell exposure to toluene and its homologues highlights the importance of substitution principle.

Down-regulation of miRNA-451a and miRNA-486-5p involved in benzene-induced inhibition on erythroid cell differentiation in vitro and in vivo.

Exposure to high-dose benzene leads to the inhibition of erythroid differentiation. However, whether lower doses of benzene exposure resemble high-dose effects in erythroid differentiation, as well as the underlying mechanisms, remains largely unknown. To identify the microRNAs (miRNAs) specifically responsible for benzene exposure and their regulatory role in erythroid differentiation, we performed miRNA microarray in CD34+ hematopoietic progenitor cells isolated from human umbilical cord blood after treatment with hydroquinone (HQ), a metabolite of benzene at concentrations of 0, 1.0, 2.5, and 5.0 µM. As a result, HQ treatment inhibited erythroid differentiation in a dose-response manner. miRNA microarray analysis revealed that miRNA-451a, miRNA-486-5p and miRNA-126-3p expression were significantly lower in HQ-treated CD34+ hematopoietic progenitor cells. In vitro studies showed that miRNA-451a and miRNA-486-5p were up-regulated during erythroid differentiation both in CD34+ hematopoietic progenitor cells and K562 cells. The increase in the percentage of benzidine-positive cells and the expression of γ-globin in K562 cells transfected with either miRNA-451a or miRNA-486-5p mimic indicated that both miRNAs played a role in the promotion of erythroid cell differentiation. Overexpression of either miRNA-451a or miRNA-486-5p attenuated the inhibitory effects on erythroid differentiation in HQ-treated K562 cells. In vivo study showed a decreasing count of peripheral red blood cell (RBC) in C57BL/6J male mice treated with aerosol benzene at concentrations of 0, 1, 5, 25 ppm (time weight average, TWA). In addition, the expression of miRNA-451a or miRNA-486-5p was negatively correlated with the concentration of benzene inhalation on erythroid toxicity of C57BL/6J mice. Particularly, the decline in miRNA-451a and miRNA-486-5p expression appeared in male chronic benzene poisoning patients, and was correlated with a constant decrease in their RBC counts over the first 3 months after being diagnosed. These findings indicate that the suppression of miRNA-451a or miRNA-486-5p might be associated with the benzene-induced perturbation of erythroid cell differentiation.

[Study on the health effects of occupational exposure to low concentrations of benzene].

Objective: The main purpose of this study was to ascertain whether (or not) exposure to benzene, toluene, xylene and ethylbenzene (BTXE) , under normal working conditions, was associated with any health effects. Methods: From January to December 2014, the workplaces concentrations of BTXE were measured of 71 enterprises in Suzhou Industrial Park. Occupational health examination were investigated on 764 employees who exposed to BTXE, as well as 4409 employees of the corresponding enterprises who unexposed to BTXE, and analyzed the data of the two groups. Results: A total of 6 monitoring sites in 3 enterprises BTXE concentrations excess of the standards, the unexposed group was under the limit of detection. The means of red blood cell count, hemoglobin, hematocrit, intermediate cell count and percentage of intermediate cells were significantly higher in exposed group than in unexposed group (P<0.05) . Conversely, platelet count was significantly lower in exposed group than in unexposed group (P<0.05) . The proportion of red blood cell volume, lymphocyte count and percentage of intermediate cells were significantly lower in exposed group than in unexposed group (P<0.05) . Both means and proportion of glutamic pyruvic transaminase and urea nitrogen were significantly higher in exposed group than in unexposed group (P<0.05) . The positive rate of protein, urine, urine red blood cell were significantly higher in exposed group than in unexposed group (P<0.05) . The abnormal rate of electrocardiogram, liver and kidney B scan were significantly higher in exposed group than in unexposed group (P<0.05) . Multivariate logistic regression analysis revealed that percentage of intermediate cells increased, urea nitrogen increased, urine protein positived, urine red blood cells positived in exposed group the OR values were 1.689, 3.291, 3.163 and 1.743 (P<0.05) . Conclusion: Occupational exposure to low concentrations of BTXE had a certain impact on the blood system and liver and kidney function of the employees, occupational health surveillance for such people should be strengthened.

Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period.

Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3mg/m3 FA for 2weeks, mimicking occupational exposure, and were monitored for another 7days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk.

Absorption of ethanol, acetone, benzene and 1,2-dichloroethane through human skin in vitro: a test of diffusion model predictions.

The overall goal of this research was to further develop and improve an existing skin diffusion model by experimentally confirming the predicted absorption rates of topically-applied volatile organic compounds (VOCs) based on their physicochemical properties, the skin surface temperature, and the wind velocity. In vitro human skin permeation of two hydrophilic solvents (acetone and ethanol) and two lipophilic solvents (benzene and 1,2-dichloroethane) was studied in Franz cells placed in a fume hood. Four doses of each (14)C-radiolabed compound were tested - 5, 10, 20, and 40µLcm(-2), corresponding to specific doses ranging in mass from 5.0 to 63mgcm(-2). The maximum percentage of radiolabel absorbed into the receptor solutions for all test conditions was 0.3%. Although the absolute absorption of each solvent increased with dose, percentage absorption decreased. This decrease was consistent with the concept of a stratum corneum deposition region, which traps small amounts of solvent in the upper skin layers, decreasing the evaporation rate. The diffusion model satisfactorily described the cumulative absorption of ethanol; however, values for the other VOCs were underpredicted in a manner related to their ability to disrupt or solubilize skin lipids. In order to more closely describe the permeation data, significant increases in the stratum corneum/water partition coefficients, Ksc, and modest changes to the diffusion coefficients, Dsc, were required. The analysis provided strong evidence for both skin swelling and barrier disruption by VOCs, even by the minute amounts absorbed under these in vitro test conditions.

Investigation into variation of endogenous metabolites in bone marrow cells and plasma in C3H/He mice exposed to benzene.

Benzene is identified as a carcinogen. Continued exposure of benzene may eventually lead to damage to the bone marrow, accompanied by pancytopenia, aplastic anemia or leukemia. This paper explores the variations of endogenous metabolites to provide possible clues for the molecular mechanism of benzene-induced hematotoxicity. Liquid chromatography coupled with time of flight-mass spectrometry (LC-TOF-MS) and principal component analysis (PCA) was applied to investigate the variation of endogenous metabolites in bone marrow cells and plasma of male C3H/He mice. The mice were injected subcutaneously with benzene (0, 300, 600 mg/day) once daily for seven days. The body weights, relative organ weights, blood parameters and bone marrow smears were also analyzed. The results indicated that benzene caused disturbances in the metabolism of oxidation of fatty acids and essential amino acids (lysine, phenylalanine and tyrosine) in bone marrow cells. Moreover, fatty acid oxidation was also disturbed in plasma and thus might be a common disturbed metabolic pathway induced by benzene in multiple organs. This study aims to investigate the underlying molecular mechanisms involved in benzene hematotoxicity, especially in bone marrow cells.

A calibrated human PBPK model for benzene inhalation with urinary bladder and bone marrow compartments.

A physiologically-based pharmacokinetic (PBPK) model of benzene inhalation based on a recent mouse model was adapted to include bone marrow (target organ) and urinary bladder compartments. Empirical data on human liver microsomal protein levels and linked CYP2E1 activities were incorporated into the model, and metabolite-specific conversion rate parameters were estimated by fitting to human biomonitoring data and adjusting for background levels of urinary metabolites. Human studies of benzene levels in blood and breath, and phenol levels in urine were used to validate the rate of human conversion of benzene to benzene oxide, and urinary benzene metabolites from Chinese benzene worker populations provided model validation for rates of human conversion of benzene to muconic acid (MA) and phenylmercapturic acid (PMA), phenol (PH), catechol (CA), hydroquinone (HQ), and benzenetriol (BT). The calibrated human model reveals that while liver microsomal protein and CYP2E1 activities are lower on average in humans compared to mice, the mouse also shows far lower rates of benzene conversion to MA and PMA, and far higher conversion of benzene to BO/PH, and of BO/PH to CA, HQ, and BT. The model also differed substantially from existing human PBPK models with respect to several metabolic rate parameters of importance to interpreting benzene metabolism and health risks in human populations associated with bone marrow doses. The model provides a new methodological paradigm focused on integrating linked human liver metabolism data and calibration using biomonitoring data, thus allowing for model uncertainty analysis and more rigorous validation.

[Epigenic modifications associated with low benzene exposure]. / Modificazioni epigenetiche nell'esposizione a basse dosi di benzene.

DNA methylation, mitochondrial DNA copy number and telomeres shortening are cellular modifications associated with an increasing number of tumors, cardiovascular and aging diseases. In our studies these modifications were evaluated in subjects occupationally exposed to low levels of benzene and in the general population. In peripheral blood lymphocytes a decrease of DNA methylation with the increase of personal benzene exposure was found, both in Alu and LINE-1 repetitive elements, and in the global DNA. Telomere length shortening in subjects exposed to traffic exhausts and an increase in mitochondrial DNA copy number correlated to benzene exposure was also found. DNA methylation measured in specimen repeats collected at intervals of 8 years decreased more markedly in exposed subjects than in controls. Our studies highlighted the association of epigenetic modifications of DNA with low benzene exposure.

[Monitoring of the occupational and environmental exposure to low doses of benzene]. / Il monitoraggio dell'esposizione occupazionale ed ambientale a basse dosi di benzene.

AIM: To verify which of the various biomarkers of internal dose of benzene can be considered reliable for biological monitoring of exposure to the low concentrations present nowadays in working and living environments. MATERIALS AND METHODS: The specific literature was analyzed to assess the reliability of the different biomarkers of internal dose. RESULTS AND CONCLUSIONS: T,t-muconic acid is a non specific biomarker for benzene, valid for exposure to concentrations up to one order of magnitude less than the threshold limit of 3250 microg/m3. S-phenylmercapturic acid (SPMA) is a reliable marker even for exposure to concentrations up to two orders below the threshold value of 3250 microg/m3, and can be considered the biomarker of choice for biological monitoring of workers exposed to benzene. Urinary benzene does not seem to have any real advantages over SPMA for monitoring occupational exposure to benzene, but it does seem to be more reliable than SPMA to assess exposure to concentrations like those present in living environments. A smoking habit influences the urinary excretion of all the described biomarkers, and for the current low levels of occupational and environmental exposure to benzene, must be taken into account when interpreting the results of biological monitoring.

Evaluation of neuropsychological symptoms and exposure to benzene, toluene and xylene among two different furniture worker groups in Izmir.

This study was conducted to determine whether there was any exposure to toluene, xylene and benzene and to assess the health impact of these solvents on workers in furniture enterprises in Karabaglar, Izmir. This cross-sectional study covered furniture enterprises in Karabaglar, Izmir. This study was comprised of an exposed group consisting of workers engaged in painting and varnishing and therefore exposed either directly or indirectly toluene, xylene and benzene in the workplace and the non-exposed group engaged in other aspects of production. While a total of 261 individuals completed questionnaires, 210 workers agreed to provide blood samples. Blood solvents levels were determined using gas chromatograph at Ege University, Intoxication Research and Application Centre. The modified EUROQUEST questionnaire was used to assess neuropsychological symptoms and neurological and general examination were performed. Occupational and exposure history, demographic and work-related information was collected. In this study of workers, blood toluene and benzene levels were found to be significantly higher among those engaged in painting and varnishing compared to those who perform other tasks. The average blood toluene and benzene concentrations among exposed workers were 6.95 times and 1.64 times respectively higher than those in the nonexposed groups. Smokers and participants who worked in excess of 8 hours/day had higher blood toluene and benzene levels. The most frequently work-related health complaints were back pain, allergies and asthma. No differences were found in the average scores in the neuropsychological symptoms questionnaire between exposed and non-exposed groups. Neurological examination of two individuals with these complaints revealed a loss of reflexes. The workers were unaware that they were being exposed to solvents at work. Tobacco smoke is a major source of internal exposure to benzene. Improving working conditions in furniture work places is a priority.

[Genetic markers of individual susceptibility and oxidative stress in subjects exposed to low dose of benzene. Preliminary data]. / Indicatori genetici di suscettibilità individuale e stress ossidativo in soggetti esposti a basse dosi di benzene. Dati preliminari.

In the present study we used AOPPs and AGE as early markers of oxidative stress in refinery oil workers. In addition we evaluated whether a genetically determined reduction in the ability to detoxify electrophilic compounds, such as that expected among individuals with glutathione S-transferase (GST) null genotypes might influence the levels of AOPPs thus increasing toxicity. The study was performed on 25 oil refinery workers and in 18 age-matched control subjects. We found a statistically significant increase of AOPPs in exposed workers with respect to controls while AGE levels were not different. Finally serum level of AOPPs and AGE were not correlated with the different GTS genotypes.

[Urinary benzene in biological monitoring of environmental exposure to low benzene concentrations]. / Il benzene urinario nel monitoraggio biologico dell'esposizione a bassi livelli ambientali.

INTRODUCTION: Conflicting opinions exist about urinary benzene (UB) as a reliable biomarker of exposure. Objective of our study is to evaluate the effect of low-level environmental exposure on UB levels. METHODS: We monitored UB excretion in 74 non-smoking non- occupationally exposed subjects; a questionnaire interview gathered information on relevant exposures during the day of monitoring. RESULTS: UB excretion was related (p < 0.05) to gender, sampling time, residence, and reported vehicular traffic, but not to passive smoking and body mass index. CONCLUSION: Our findings support the use of unmetabolized UB as a specific and sensitive biomarker of low-level exposure to benzene.

Benzene-induced histopathological changes and germ cell population dynamics in testes of Sprague Dawley rats.

Benzene has been considered as an occupational hematotoxin and leukemogen. The present study was conducted to determine the effects of oral administration of benzene on reproductive organs and testicular spermatogenesis in rats. Adult rats were divided into three weight matched groups (Gr. I-III) containing 6 each. Gr. I rats received vehicle only and served as control. Rats in Gr. II and III were fed orally with 0.5 and 1 ml kg(-1) dose of benzene for 14 and 9 days, respectively and autopsy was done on 15th and 10th day. Food and water intake and gross behavioral changes were recorded daily during the entire treatment. Results showed no significant change in reproductive organ weights viz. testis, epididymis and ventral prostate in benzene-treated (0.5 or 1 ml kg(-1)) rats than that in controls. But, caused a significant decrease (p < 0.005) in weights of seminal vesicles in rats treated with both 0.5 and 1 ml kg(-1) doses compared to control. In contrast, at higher dose (1 ml kg(-1)) of benzene, significant (p < 0.001) decline in body weight and 100% mortality was observed on day 10 of autopsy. In treated rats, testicular cytotoxicity was marked by multinucleated giant cells formation, cytoplasmic vacuolization, pyknosis of nuclei, chromatolysis, desquamation and dissolution of germ cells in tubular lumen. The quantitative analysis of spermatogenesis showed a significant (p < 0.001) decrease in number ofA-spermatogonia (in 1 ml kg(-1) dose only), primary spermatocytes (non-pachytene and pachytene) and spermatids (round and elongated) in treated as compared to control rats. The diameters of testicular tubules and Leydig cells nuclei were also significantly (p < 0.001) reduced in treated rats. A steady loss in food and water intake recorded and signs of ill health were observed in treated (0.5 or 1 ml kg(-1)) rats. Results of the study indicated antitesticular lantispermatogenic effects of benzene at 0.5 and 1 ml kg(-1) dose in rats.

Structure-Based Discovery of Pyrimidine Aminobenzene Derivatives as Potent Oral Reversal Agents against P-gp- and BCRP-Mediated Multidrug Resistance.

Overexpression of ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), is an important factor leading to multidrug resistance (MDR) in cancer treatments. Three subclasses of dual inhibitors of P-gp and BCRP were designed based on the active moieties of BCRP inhibitors, tyrosine kinase inhibitors, and P-gp inhibitors, of which compound 21 possessed low cytotoxicity, high reversal potency, and good lipid distribution coefficient. 21 also increased the accumulation of Adriamycin (ADM) and Mitoxantrone (MX), blocked Rh123 efflux, and made no change in the protein expression of P-gp and BCRP. Importantly, coadministration of 21 can significantly improve the oral bioavailability of paclitaxel (PTX). It was also demonstrated that 21 significantly inhibited the growth of K562/A02 xenograft tumors by increasing the sensitivity of ADM in vivo. In summary, 21 has the potential to overcome MDR caused by P-gp and BCRP and to improve the oral bioavailability of PTX.

Advances in understanding benzene health effects and susceptibility.

Benzene is a ubiquitous chemical in our environment that causes acute leukemia and probably other hematological cancers. Evidence for an association with childhood leukemia is growing. Exposure to benzene can lead to multiple alterations that contribute to the leukemogenic process, indicating a multimodal mechanism of action. Research is needed to elucidate the different roles of multiple metabolites in benzene toxicity and the pathways that lead to their formation. Studies to date have identified a number of polymorphisms in candidate genes that confer susceptibility to benzene hematotoxicity. However, a genome-wide study is needed to truly assess the role of genetic variation in susceptibility. Benzene affects the blood-forming system at low levels of occupational exposure, and there is no evidence of a threshold. There is probably no safe level of exposure to benzene, and all exposures constitute some risk in a linear, if not supralinear, and additive fashion.

A Bayesian network model for biomarker-based dose response.

A Bayesian network model was developed to integrate diverse types of data to conduct an exposure-dose-response assessment for benzene-induced acute myeloid leukemia (AML). The network approach was used to evaluate and compare individual biomarkers and quantitatively link the biomarkers along the exposure-disease continuum. The network was used to perform the biomarker-based dose-response analysis, and various other approaches to the dose-response analysis were conducted for comparison. The network-derived benchmark concentration was approximately an order of magnitude lower than that from the usual exposure concentration versus response approach, which suggests that the presence of more information in the low-dose region (where changes in biomarkers are detectable but effects on AML mortality are not) helps inform the description of the AML response at lower exposures. This work provides a quantitative approach for linking changes in biomarkers of effect both to exposure information and to changes in disease response. Such linkage can provide a scientifically valid point of departure that incorporates precursor dose-response information without being dependent on the difficult issue of a definition of adversity for precursors.

Benzene inhalation by parts washers: new estimates based on measures of occupational exposure to solvent coaromatics.

Questions persist regarding assessment of workers' exposures to products containing low levels of benzene, such as mineral spirit solvent (MSS). This study summarizes previously unpublished data for parts-washing activities, and evaluates potential daily and lifetime cumulative benzene exposures incurred by workers who used historical and current formulations of a recycled mineral spirits solvent in manual parts washers. Measured benzene concentrations in historical samples from parts-washing operations were frequently below analytical detection limits. To better assess benzene exposure among these workers, air-to-solvent concentration ratios measured for toluene, ethylbenzene, and xylenes (TEX) were used to predict those for benzene based on a statistical model, conditional on physical-chemical theory supported by new thermodynamic calculations of TEX and benzene activity coefficients in a modeled MSS-type solvent. Using probabilistic methods, the distributions of benzene concentrations were then combined with distributions of other exposure parameters to estimate eight-hour time-weighted average (TWA) exposure concentration distributions and corresponding daily respiratory dose distributions for workers using these solvents in parts washers. The estimated 50th (95th) percentile of the daily respiratory dose and corresponding eight-hour TWA air concentration for workers performing parts washing are 0.079 (0.77) mg and 0.0030 (0.028) parts per million by volume (ppm) for historical solvent, and 0.020 (0.20) mg and 0.00078 (0.0075) ppm for current solvent, respectively. Both 95th percentile eight-hour TWA respiratory exposure estimates for solvent formulations are less than 10% of the current Occupational Safety and Health Administration permissible exposure limit of 1.0 ppm for benzene.

In utero and acute exposure to benzene: investigation of DNA double-strand breaks and DNA recombination in mice.

Benzene, a ubiquitous pollutant, has been identified as a human leukemogen and early exposure to environmental carcinogens such as benzene has been linked to childhood leukemia. It is known that genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which can initiate DNA recombinational repair mechanisms. In this study we investigated the induction of micronuclei, the formation of gamma-H2A.X as a marker of DNA DSBs, and the induction of somatic DNA recombination events in hematopoietic tissue from pKZ1 transgenic mice exposed acutely or in utero to benzene. Adult male C57Bl/6N mice were treated with a single i.p. injection of benzene, and timed-pregnant females pKZ1 were treated with daily i.p. injections of 200 mg/kg or 400 mg/kg benzene through gestational days 7-15. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice exposed in utero. Immunoblotting techniques did not detect benzene-induced increases in the formation of gamma-H2A.X in bone marrow cells of adult male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells after specific time-point exposures. Finally, no recombination events were detected in adult pKZ1 mouse tissue; however, in post-natal day 9 pups in utero exposure to 400 mg/kg of benzene caused a trend towards increasing recombination frequency although this did not reach statistical significance. These results demonstrate that in utero exposure increases the frequency of micronuclei and DNA recombination events in hematopoietic tissue of fetal and post-natal mice and may be an initiating event in the etiology of childhood leukemias. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias.