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Background: Impairment of the hematopoietic system is one of the primary adverse health effects from exposure to benzene. We previously have shown that exposure to benzene at low levels (<1 ppm) affects the blood forming system and that these effects were proportionally stronger at lower versus higher levels of benzene exposure. This observation is potentially explained by saturation of enzymatic systems., Methods: Here we extend these analyses by detailed modeling of the exposure response association of benzene and its major metabolites (i.e. catechol, muconic acid, phenol, and hydroquinone) on peripheral white blood cell (WBC) counts and its major cell-subtypes (i.e. granulocytes, lymphocytes, and monocytes) using two previously published cross-sectional studies among occupationally exposed Chinese workers., Results: Supra-linear exposure response associations were observed between air benzene concentrations (range ∼ 0.1 - 100 ppm) and WBC counts and its cell-subtypes, with a larger than proportional decrease in cell counts at lower than at higher levels of benzene exposure. The hematotoxicity associations were largely similar in shape when the analyses were repeated with benzene urinary metabolites suggesting that enzymatic saturation is not a full explanation of the observed non-linearity with WBC endpoints., Discussion: We hypothesize that the flattening of the exposure response curve especially at higher benzene exposure levels may reflect a response by the bone marrow to maintain hematopoietic homeostasis. Toxicity to the bone marrow and an induced hyper-proliferative response could both contribute to risk of subsequently developing a hematopoietic malignancy. Additional work is needed to explore this hypothesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The efficiency of mine excavation has been significantly enhanced by continuing improvements in tunneling capabilities; however, this has also resulted in serious environmental pollution and greater safety risks for workers. To ensure safe production, the focus of this study is on the effect of varying the air pressure and the distance between the air pressure cylinder and the side wall settings on dust dispersion behavior and dust control in excavated tunnels. We also investigated temporal-spatial dust diffusion rules in tunnels by combining numerical simulation data with field measurement results. Through further analysis, when the pressure air volume and the exhaust air volume are both equal to 250 m 3 /min, the dust diffusion distance could be fitted as: [Formula: see text]. When the exhaust air volume is equal to 250 m 3 /min, dust control effects were improved as the pressure air volume decreased, becoming optimal when the pressure air volume dropped to 150 m 3 /min. Under these conditions, areas of high dust pollution were contained within 16 m of the cutting face, and the dust diffusion distance satisfied the formula: [Formula: see text]. When the pressure air volume is fixed, the change of the distance between the pressure air cylinder and the side wall has little effect on the dust diffusion. When the distance is 1.5 m, the dust control effect is the best, and the high dust pollution area is controlled within 14 m of the cutting surface. This alleviated dust pollution to a certain degree, thereby enhancing the air quality and ensuring safer production. This study provides a new understanding of the environmentally sustainable development of tunnels and is of great significance for clean production., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Human brucellosis is caused by Brucella species and remains a major burden in both human and domesticated animal populations, especially in Inner Mongolia, China. The aims of this study were to analyze the spatiotemporal trends in human brucellosis in Inner Mongolia during 2010 to 2015, to explore the factors affecting the incidence of brucellosis. The results showed that the annual incidence was 29.68-77.67 per 100,000, and peaked from March to June. The majority of human brucellosis was male farmers and herdsmen, aged 40-59 years. The high-risk areas were mainly Xilin Gol League and Hulunbeier City. The incidence of human brucellosis in Inner Mongolia decreased during 2010 to 2015, although the middle and eastern regions were still high-risk areas. The regions with larger number of sheep and cattle, lower GDP per capita, less number of hospital beds, higher wind speed, lower mean temperature more likely to become high-risk areas of human brucellosis., (© 2021. The Author(s).)

Benzene is a recognized hematotoxin and leukemogen; however, its mechanism of action in humans remain unclear. To provide insight into the processes underlying benzene hematotoxicity, we performed high-resolution metabolomic profiling of plasma collected from a cross-sectional study of 33 healthy workers exposed to benzene (median 8-h time-weighted average exposure; 20 ppma), and 25 unexposed controls in Shanghai, China. Metabolic features associated with benzene were identified using a metabolome-wide association study (MWAS) that tested for the relationship between feature intensity and benzene exposure. MWAS identified 478 mass spectral features associated with benzene exposure at false discovery rate < 20%. Comparison to a list of 13 known benzene metabolites and metabolites predicted using a multi-component biotransformation algorithm showed five metabolites were detected, which included the known metabolites phenol and benzene diolepoxide. Metabolic pathway enrichment identified 41 pathways associated with benzene exposure, with altered pathways including carnitine shuttle, fatty acid metabolism, sulfur amino acid metabolism, glycolysis, gluconeogenesis and branched chain amino acid metabolism. These results suggest disruption to fatty acid uptake, energy metabolism and increased oxidative stress, and point towards pathways related to mitochondrial dysfunction, which has previously been linked to benzene exposure in animal models and human studies. Taken together, these results suggest benzene exposure is associated with disruption of mitochondrial pathways, and provide promising, systems biology biomarkers for risk assessment of benzene-induced hematotoxicity in humans., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Iron and steel industry workers are exposed to high levels of inhalable dust particles that contain various elements, including metals, and cause occupational lung diseases. We aim to assess the relationship between occupational dust exposure, systemic inflammation, and spirometric decline in a cohort of Chinese iron and steel workers., Methods: We studied 7513 workers who participated in a Health Surveillance program at Wugang Institute for Occupational Health between 2008 and 2017. Time-weighted exposure intensity (TWEI) of dust was quantified based on self-reported dust exposure history, the experience of occupational hygienists, and historical data of dust exposure for workers with certain job titles. A linear mixed-effects model was used for association analyses., Results: The average annual change of lung function was - 50.78 ml/year in forced expiratory volume in 1 s (FEV1) and - 34.36 ml/year in forced vital capacity (FVC) in males, and - 39.06 ml/year in FEV1 and - 26.66 ml/year in FVC in females. Higher TWEI prior to baseline was associated with lower longitudinal measurements of FEV1 and FVC but not with their decline rates. Higher WBC and its differential at baseline were associated with lower longitudinal measurements and a more rapid decline of FEV1 and FVC in a dose-dependent monotonically increasing manner. Moreover, the increase of WBC and its differential post-baseline was also associated with a more rapid decline of FEV1 and FVC., Conclusions: Our findings support the important role of systemic inflammation in affecting the temporal change of lung function in iron and steel industry workers., (© 2021. The Author(s).)

Background: Previously, using microarrays and mRNA-Sequencing (mRNA-Seq) we found that occupational exposure to a range of benzene levels perturbed gene expression in peripheral blood mononuclear cells., Objectives: In the current study, we sought to identify gene expression biomarkers predictive of benzene exposure below 1 part per million (ppm), the occupational standard in the U.S., Methods: First, we used the nCounter platform to validate altered expression of 30 genes in 33 unexposed controls and 57 subjects exposed to benzene (<1 to ≥5 ppm). Second, we used SuperLearner (SL) to identify a minimal number of genes for which altered expression could predict <1 ppm benzene exposure, in 44 subjects with a mean air benzene level of 0.55±0.248 ppm (minimum 0.203ppm)., Results: nCounter and microarray expression levels were highly correlated (coefficients >0.7, p<0.05) for 26 microarray-selected genes. nCounter and mRNA-Seq levels were poorly correlated for 4 mRNA-Seq-selected genes. Using negative binomial regression with adjustment for covariates and multiple testing, we confirmed differential expression of 23 microarray-selected genes in the entire benzene-exposed group, and 27 genes in the <1 ppm-exposed subgroup, compared with the control group. Using SL, we identified 3 pairs of genes that could predict <1 ppm benzene exposure with cross-validated AUC estimates >0.9 (p<0.0001) and were not predictive of other exposures (nickel, arsenic, smoking, stress). The predictive gene pairs are PRG2/CLEC5A, NFKBI/CLEC5A, and ACSL1/CLEC5A. They play roles in innate immunity and inflammatory responses., Conclusions: Using nCounter and SL, we validated the altered expression of multiple mRNAs by benzene and identified gene pairs predictive of exposure to benzene at levels below the US occupational standard of 1ppm., Competing Interests: G.L. has received funds from the American Petroleum Institute for consulting on benzene-related health research. S.M.R. has received consulting and expert testimony fees from law firms representing plaintiffs’ cases involving exposure to benzene and has received research support from the American Petroleum Institute and the American Chemistry Council. M.T.S. has received consulting and expert testimony fees from law firms representing both plaintiffs and defendants in cases involving exposure to benzene. The other authors declare that they have no actual or potential competing financial interests. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Exposure to benzene, a known leukemogen and probable lymphomagen, has been demonstrated to result in oxidative stress, which has previously been associated with altered telomere length (TL). TL specifically has been associated with several health outcomes in epidemiologic studies, including cancer risk, and has been demonstrated to be altered following exposure to a variety of chemical agents. To evaluate the association between benzene exposure and TL, we measured TL by monochrome multiplex quantitative PCR in 43 workers exposed to high levels of benzene and 43 age and sex-matched unexposed workers in Shanghai, China. Benzene exposure levels were monitored using organic vapor passive dosimetry badges before phlebotomy. The median benzene exposure level in exposed workers was 31 ppm. The mean TL in controls, workers exposed to levels of benzene below the median (≤31 ppm), and above the median (>31 ppm) was 1.26 ± 0.17, 1.25 ± 0.16, and 1.37 ± 0.23, respectively. Mean TL was significantly elevated in workers exposed to >31 ppm of benzene compared with controls (P = 0.03). Our findings provide evidence that high levels of occupational benzene exposure are associated with TL. Environ., (Published [2014]. This article is a U.S. Government work and is in the public domain in the USA.)

Benzene exposure causes acute myeloid leukemia and hematotoxicity, shown as suppression of mature blood and myeloid progenitor cell numbers. As the leukemia-related aneuploidies monosomy 7 and trisomy 8 previously had been detected in the mature peripheral blood cells of exposed workers, we hypothesized that benzene could cause leukemia through the induction of these aneuploidies in hematopoietic stem and progenitor cells. We measured loss and gain of chromosomes 7 and 8 by fluorescence in situ hybridization in interphase colony-forming unit-granulocyte-macrophage (CFU-GM) cells cultured from otherwise healthy benzene-exposed (n=28) and unexposed (n=14) workers. CFU-GM monosomy 7 and 8 levels (but not trisomy) were significantly increased in subjects exposed to benzene overall, compared with levels in the control subjects (P=0.0055 and P=0.0034, respectively). Levels of monosomy 7 and 8 were significantly increased in subjects exposed to <10 p.p.m. (20%, P=0.0419 and 28%, P=0.0056, respectively) and ≥ 10 p.p.m. (48%, P=0.0045 and 32%, 0.0354) benzene, compared with controls, and significant exposure-response trends were detected (P(trend)=0.0033 and 0.0057). These data show that monosomies 7 and 8 are produced in a dose-dependent manner in the blood progenitor cells of workers exposed to benzene, and may be mechanistically relevant biomarkers of early effect for benzene and other leukemogens.

Evidence suggests that de novo, therapy-related and benzene-induced acute myeloid leukemias (AML) occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes has been detected in benzene-related leukemia patients as well as in healthy benzene-exposed workers, suggesting that aneuploidy precedes and may be a potential mechanism underlying benzene-induced leukemia. Here, we analyzed the peripheral blood lymphocytes of 47 exposed workers and 27 unexposed controls using a novel OctoChrome fluorescence in situ hybridization (FISH) technique that simultaneously detects aneuploidy in all 24 chromosomes. Through this chromosome-wide aneuploidy study (CWAS) approach, we found heterogeneity in the monosomy and trisomy rates of the 22 autosomes when plotted against continuous benzene exposure. In addition, statistically significant, chromosome-specific increases in the rates of monosomy [5, 6, 7, 10, 16 and 19] and trisomy [5, 6, 7, 8, 10, 14, 16, 21 and 22] were found to be dose dependently associated with benzene exposure. Furthermore, significantly higher rates of monosomy and trisomy were observed in a priori defined 'susceptible' chromosome sets compared with all other chromosomes. Together, these findings confirm that benzene exposure is associated with specific chromosomal aneuploidies in hematopoietic cells, which suggests that such aneuploidies may play roles in benzene-induced leukemogenesis.

We previously reported evidence that humans metabolize benzene via two enzymes, including a hitherto unrecognized high-affinity enzyme that was responsible for an estimated 73% of total urinary metabolites [sum of phenol (PH), hydroquinone (HQ), catechol (CA), E,E-muconic acid (MA), and S-phenylmercapturic acid (SPMA)] in nonsmoking females exposed to benzene at sub-saturating (ppb) air concentrations. Here, we used the same Michaelis-Menten-like kinetic models to individually analyze urinary levels of PH, HQ, CA and MA from 263 nonsmoking Chinese women (179 benzene-exposed workers and 84 control workers) with estimated benzene air concentrations ranging from less than 0.001-299 ppm. One model depicted benzene metabolism as a single enzymatic process (1-enzyme model) and the other as two enzymatic processes which competed for access to benzene (2-enzyme model). We evaluated model fits based upon the difference in values of Akaike's Information Criterion (DeltaAIC), and we gauged the weights of evidence favoring the two models based upon the associated Akaike weights and Evidence Ratios. For each metabolite, the 2-enzyme model provided a better fit than the 1-enzyme model with DeltaAIC values decreasing in the order 9.511 for MA, 7.379 for PH, 1.417 for CA, and 0.193 for HQ. The corresponding weights of evidence favoring the 2-enzyme model (Evidence Ratios) were: 116.2:1 for MA, 40.0:1 for PH, 2.0:1 for CA and 1.1:1 for HQ. These results indicate that our earlier findings from models of total metabolites were driven largely by MA, representing the ring-opening pathway, and by PH, representing the ring-hydroxylation pathway. The predicted percentage of benzene metabolized by the putative high-affinity enzyme at an air concentration of 0.001 ppm was 88% based upon urinary MA and was 80% based upon urinary PH. As benzene concentrations increased, the respective percentages of benzene metabolized to MA and PH by the high-affinity enzyme decreased successively to 66 and 77% at 0.1 ppm, 20 and 58% at 1 ppm, and 2.7 and 17% at 10 ppm. This indicates that the putative high-affinity enzyme was active primarily below 1 ppm and favored the ring-opening pathway., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Benzene is an established cause of leukemia, and possibly lymphoma, in humans, but the underlying molecular pathways remain largely undetermined. We used two microarray platforms to identify global gene expression changes associated with well-characterized occupational benzene exposure in the peripheral blood mononuclear cells (PBMC) of a population of shoe-factory workers. Differential expression of 2692 genes (Affymetrix) and 1828 genes (Illumina) was found and the concordance was 50% (based on an average fold-change > or =1.3 from the two platforms), with similar expression ratios among the concordant genes. Four genes (CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were among the top 100 genes identified by both platforms in the current study. Gene ontology analysis showed overrepresentation of genes involved in apoptosis among the concordant genes while pathway analysis identified pathways related to lipid metabolism. The two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.

Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. Benzene metabolites can produce reactive oxidative species. Mitochondrial DNA (mtDNA) copy number may be increased in response to oxidative stress to compensate for damaged mitochondria. We carried out a cross-sectional study of 40 benzene-exposed workers and 40 controls to evaluate the association between benzene exposure and mtDNA copy number. Copy number of mtDNA in leukocyte DNA was determined by real-time PCR. Compared with controls, the copy number of mtDNA increased by 4% and by 15% in workers exposed to < or =10 ppm (n = 20) and >10 ppm (n = 20) benzene, respectively. After adjusting for recent infection, the factor that was significantly correlated with mtDNA, the increase of mtDNA was statistically significant in the high exposed group (P = 0.016) with a significant linear trend (P = 0.024). To our best knowledge, this is the first report that benzene exposure was associated with increased mitochondria DNA copy number. Benzene exposure may induce mtDNA amplification, possibly in response to oxidative stress caused by benzene. The finding needs to be replicated by other studies.

As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/microg DNA, as opposed to 55% of 20 controls (P = .0063 by Fisher's exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.

Epidemiological studies show that benzene exposure is associated with an increased incidence of leukemia and perhaps lymphoma. Chromosomal rearrangements are common in these hematopoietic diseases. Translocation t(14;18), the long-arm deletion of chromosome 6 [del(6q)], and trisomy 12 are frequently observed in lymphoma patients. Rearrangements of the MLL gene located on chromosome 11q23, such as t(4;11) and t(6;11), are common in therapy-related leukemias resulting from treatment with topoisomerase II inhibiting drugs. To examine numerical and structural changes in these chromosomes (2, 4, 6, 11, 12, 14, and 18), fluorescence in situ hybridization (FISH) was employed on metaphase spreads from workers exposed to benzene (n = 43) and matched controls (n = 44) from Shanghai, China. Aneuploidy (both monosomy and trisomy) of all seven chromosomes was increased by benzene exposure. Benzene also induced del(6q) in a dose-dependent manner (P(trend) = 0.0002). Interestingly, translocations between chromosomes 14 and 18, t(14;18), known to be associated with follicular non-Hodgkin lymphoma, were increased in the highly exposed workers (P < 0.001). On the other hand, translocations between chromosome 11 and other partner chromosomes that are found in therapy-induced leukemias were not increased. These data add weight to the notion that benzene can induce t(14;18) and del(6q) found in lymphoma, but do not support the idea that benzene induces t(4;11) or t(6;11). However, they do not rule out the possibility that other rearrangements of the MLL gene at chromosome 11q23 may be induced by benzene., ((c) 2007 Wiley-Liss, Inc.)

Benzene is a recognized haematotoxin and leukaemogen, but its mechanism of action and the role of genetic susceptibility are still unclear. Cytochrome P450 2E1 (CYP2E1) and myeloperoxidase (MPO) are involved in benzene activation; and NAD (P)H:quinine oxidoreductase 1 (NQO1), glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase mu 1 (GSTM1) participate in benzene detoxification. The common, well-studied single-nucleotide polymorphisms (SNPs) were analysed in these genes drawn from the toxicant-metabolizing pathways. A total of 100 workers with chronic benzene poisoning (CBP) and 90 controls were enrolled in China. There was a 2.82-fold (95% CI = 1.42-5.58) increased risk of CBP in the subjects with the NQO1 609C > T mutation genotype (T/T) compared with those carrying heterozygous (C/T) and wild-type (C/C). The subjects with the GSTT1 null genotype had a 1.91-fold (95% CI = 1.05-3.45) increased risk of CBP compared with those with GSTT1 non-null genotype. There was no association of CYP2E1 and MPO genotype with CBP. A three genes' interaction showed that there was a 20.41-fold (95% CI = 3.79-111.11) increased risk of CBP in subjects with the NQO1 609C > T T/T genotype and with the GSTT1 null genotype and the GSTM1 null genotype compared with those carrying the NQO1 609C > T C/T and C/C genotype, GSTT1 non-null genotype, and GSTM1 non-null genotype. The study provides evidence of an association of a gene-gene interaction with the risk of CBP.

Objectives: The aim of this investigation was to use activated carbon cloth (ACC) patches to study the probability and extent of dermal exposure to benzene and toluene in a shoe factory., Methods: Inhalation and dermal exposure loading were measured simultaneously in 70 subjects on multiple days resulting in 113 observations. Dermal exposure loading was assessed by ACC patches attached to likely exposed skin areas (e.g. the palm of the hand and abdomen). A control patch at the chest and an organic vapor monitor (OVM) were used to adjust the hand and abdomen patches for the contribution from the air through passive absorption of benzene and toluene on the ACC patches. Systemic exposure was assessed by quantification of unmetabolized benzene (UBz) and toluene (UTol) in urine., Results: Mean air concentrations for the study population were 1.5 and 7.5 ppm for benzene and toluene, respectively. Iterative regression analyses between the control patch, OVM and the dermal patches showed that only a small proportion of the ACC patches at the hand had likely benzene (n = 4; mean 133 microg cm(-2) h(-1)) or toluene (n = 5; mean 256 microg cm(-2) h(-1)) contamination. Positive patches were exclusively observed among subjects performing the task of gluing. Significant dermal exposure loading to the abdomen was detected only for toluene (n = 2; mean 235 microg cm(-2) h(-1)). No relation was found between having a positive hand or abdomen ACC patch and UBz or UTol levels. In contrast a strong association was found between air levels of benzene (p = 0.0016) and toluene (p < 0.0001) and their respective urinary levels., Conclusions: ACC patches are shown to be a useful technique for quantifying the probability of dermal exposure to organic solvents and to provide estimates of the potential contribution of the dermal pathway to systemic exposure. Using ACC patches we show that dermal exposure to benzene and toluene in a shoe manufacturing factory is probably rare, and when it occurred exposures were relatively low and did not significantly contribute to systemic exposure.

Although the toxicity of benzene has been linked to its metabolism, the dose-related production of metabolites is not well understood in humans, particularly at low levels of exposure. We investigated unmetabolized benzene in urine (UBz) and all major urinary metabolites [phenol (PH), E,E-muconic acid (MA), hydroquinone (HQ) and catechol (CA)] as well as the minor metabolite, S-phenylmercapturic acid (SPMA), in 250 benzene-exposed workers and 139 control workers in Tianjin, China. Median levels of benzene exposure were approximately 1.2 p.p.m. for exposed workers (interquartile range: 0.53-3.34 p.p.m.) and 0.004 p.p.m. for control workers (interquartile range: 0.002-0.007 p.p.m.). (Exposures of control workers to benzene were predicted from levels of benzene in their urine.) Metabolite production was investigated among groups of 30 workers aggregated by their benzene exposures. We found that the urine concentration of each metabolite was consistently elevated when the group's median benzene exposure was at or above the following air concentrations: 0.2 p.p.m. for MA and SPMA, 0.5 p.p.m. for PH and HQ, and 2 p.p.m. for CA. Dose-related production of the four major metabolites and total metabolites (micromol/l/p.p.m. benzene) declined between 2.5 and 26-fold as group median benzene exposures increased between 0.027 and 15.4 p.p.m. Reductions in metabolite production were most pronounced for CA and PH<1 p.p.m., indicating that metabolism favored production of the toxic metabolites, HQ and MA, at low exposures.

This study expands a previous study of NAT2 polymorphisms and bladder cancer in male subjects occupationally exposed only to benzidine. The combined analysis of 68 cases and 107 controls from a cohort of production workers in China exposed to benzidine included 30 new cases and 67 controls not previously studied. NAT2 enzymatic activity phenotype was characterized by measuring urinary caffeine metabolite ratios. PCR-based methods identified genotypes for NAT2, NAT1 and GSTM1. NAT2 phenotype and genotype data were consistent. A protective association was observed for the slow NAT2 genotype (bladder cancer OR = 0.3; 95% CI = 0.1 = 1.0) after adjustment for cumulative benzidine exposure and lifetime smoking. Individuals carrying NAT1wt/*10 and NAT1*10/*10 showed higher relative risks of bladder cancer (OR = 2.8, 95% CI = 0.8-10.1 and OR = 2.2, 95% CI = 0.6-8.3, respectively). No association was found between GSTM1 null and bladder cancer. A metaanalysis risk estimate of case-control studies of NAT2 acetylation and bladder cancer in Asian populations without occupational arylamine exposures showed an increased risk for slow acetylators. The lower limit of the confidence interval (OR = 1.4; 95% CI = 1.0-2.0) approximated the upper confidence interval for the estimate obtained in our analysis. These results support the earlier finding of a protective association between slow acetylation and bladder cancer in benzidine-exposed workers, in contrast to its established link as a risk factor for bladder cancer in people exposed to 2-naphthylamine and 4-aminobiphenyl. Study findings suggest the existence of key differences in the metabolism of mono- and diarylamines., (Published 2005 Wiley-Liss, Inc.)

Benzene is an important industrial chemical and environmental contaminant that causes leukemia. To obtain mechanistic insight into benzene's mechanism of action, we examined the impact of benzene on the human serum proteome in a study of exposed healthy shoe-factory workers and unexposed controls. Two sequential studies were performed, each using sera from 10 workers exposed to benzene (overall mean benzene air level >30 ppm) and 10 controls. Serum samples were subjected to anion-exchange fractionation and bound to three types of ProteinChip arrays (Ciphergen Biosystems, Fremont, CA) [hydrophobic (H50), metal affinity (IMAC3-Cu), and cation exchange (WCX2)]. Protein-expression patterns were detected by surface-enhanced laser desorption/ionization (SELDI)-TOF MS. Three proteins (4.1, 7.7, and 9.3 kDa) were consistently down-regulated in exposed compared with control subjects in both studies. All proteins were highly inversely correlated with individual estimates of benzene exposure (r > 0.75). The 7.7- and 9.3-kDa proteins were subsequently identified as platelet factor (PF)4 and connective tissue activating peptide (CTAP)-III. Initial proteomic results for PF4 and CTAP-III were subsequently confirmed in a single experiment using a ProteinChip-array-based immunoassay(Ciphergen Biosystems). The altered expression of the platelet-derived CXC-chemokines (40% and 63% for PF4 and CTAP-III, respectively) could not be explained by changes in absolute platelet counts. Thus, SELDI-TOF analysis of a limited number of exposed and unexposed subjects revealed that lowered expression of PF4 and CTAP-III proteins is a potential biomarker of benzene's early biologic effects and may play a role in the immunosuppressive effects of benzene.

Benzene is a recognized hematotoxin and leukemogen but its mechanism of action and the role of genetic susceptibility are still unclear. Cytokines, chemokines, and cellular adhesion molecules are soluble proteins that play an important regulatory role in hematopoiesis. We therefore hypothesized that variation in these genes could influence benzene-induced hematotoxicity. We analyzed common, well-studied single-nucleotide polymorphisms (SNPs) in 20 candidate genes drawn from these pathways in a study of 250 workers exposed to benzene and 140 unexposed controls in China. After accounting for multiple comparisons, SNPs in five genes were associated with a statistically significant decrease in total WBC counts among exposed workers [IL-1A (-889C>T), IL-4 (-1098T>G), IL-10 (-819T>C), IL-12A (8685G>A), and VCAM1 (-1591T>C)], and one SNP [CSF3 (Ex4-165C>T)] was associated with an increase in WBC counts. The adhesion molecule VCAM1 variant was particularly noteworthy as it was associated with a decrease in B cells, natural killer cells, CD4+ T cells, and monocytes. Further, VCAM1 (-1591T>C) and CSF3 (Ex4-165C>T) were associated, respectively, with decreased (P = 0.041) and increased (P = 0.076) CFU-GEMM progenitor cell colony formation in 29 benzene-exposed workers. This is the first report to provide evidence that SNPs in genes that regulate hematopoiesis influence benzene-induced hematotoxicity.

Benzene is an industrial chemical and component of gasoline that is an established cause of leukemia. To better understand the risk benzene poses, we examined the effect of benzene exposure on peripheral blood mononuclear cell (PBMC) gene expression in a population of shoe-factory workers with well-characterized occupational exposures using microarrays and real-time polymerase chain reaction (PCR). PBMC RNA was stabilized in the field and analyzed using a comprehensive human array, the U133A/B Affymetrix GeneChip set. A matched analysis of six exposed-control pairs was performed. A combination of robust multiarray analysis and ordering of genes using paired t-statistics, along with bootstrapping to control for a 5% familywise error rate, was used to identify differentially expressed genes in a global analysis. This resulted in a set of 29 known genes being identified that were highly likely to be differentially expressed. We also repeated these analyses on a smaller subset of 508 cytokine probe sets and found that the expression of 19 known cytokine genes was significantly different between the exposed and the control subjects. Six genes were selected for confirmation by real-time PCR, and of these, CXCL16, ZNF331, JUN, and PF4 were the most significantly affected by benzene exposure, a finding that was confirmed in a larger data set from 28 subjects. The altered expression was not caused by changes in the makeup of the PBMC fraction. Thus, microarray analysis along with real-time PCR confirmation reveals that altered expressions of CXCL16, ZNF331, JUN, and PF4 are potential biomarkers of benzene exposure.

Benzene is an established human leukemogen. The mechanism of benzene-induced leukemogenesis, however, remains unclear, but chromosomal damage is thought to play a critical role. We previously reported that the loss of chromosomes 5 and 7 (monosomy 5 and 7) and the gain of chromosomes 8 and 21 (trisomy 8 and 21) are significantly increased in benzene-exposed workers in comparison to matched controls. To determine if selective effects of benzene can occur, we employed three-color painting on an 8-square slide to screen numerical changes in all 24 human chromosomes (OctoChrome FISH) in a pilot study of 11 subjects (6 exposed to >5 ppm benzene and 5 age- and sex-matched controls). The effects of benzene on each chromosome were assessed as the incidence rate ratio (IRR) from a Poisson regression model with the strongest effects being reflected by the highest IRR values. Monosomy of chromosomes 5, 6, 7 and 10 had the highest IRRs and statistical significance in this preliminary study (IRR>2.5, p<0.01). On the other hand, the monosomy levels of six other chromosomes (1, 4, 9, 11, 22 and Y) were unchanged in the exposed workers with IRRs close to 1.0. Similarly, selective effects were also observed on trisomy induction with chromosomes 8, 9, 17, 21 and 22 (IRR>2.5, p<0.01). These results suggest that benzene has the capability of producing selective effects on certain chromosomes, which is supported by our in vitro findings showing that chromosomes 5 and 7 are more sensitive to loss than other chromosomes following exposure to benzene metabolites. We are currently investigating potential mechanisms for this induction of selective aneuploidy.

This report is part of an extensive biomarker study conducted in a Chinese occupational population with benzene exposures ranging from 0.06 to 122 ppm (median exposure of 3.2 ppm). All urinary benzene metabolites measured in this study were significantly elevated after exposure to benzene at or above 5 ppm. Among these metabolites, however, only S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) showed a significant exposure-response trend over the exposure range from 0 to 1 ppm (for S-PMA, p<0.0001 and for t,t-MA, p=0.006). For benzene exposure monitoring, both S-PMA and t,t-MA were judged to be good and sensitive markers, which detected benzene exposure at around 0.1 and 1 ppm, respectively. Polymorphisms of the metabolic genes, including CYP2E1, quinone oxidoreductase (NQO1), GSTT1, and myeloperoxidase (MPO), were identified and did not show significant effects on the formation of metabolites, except GSTT1 on S-PMA. The production rate of S-PMA from benzene in exposed workers with GSTT1 null alleles (24.72+/-32.48 microg/g creatinine/ppm benzene) was significantly lower than that in subjects with the wild type of GSTT1 (59.84+/-47.66 microg/g creatinine/ppm benzene, p<0.0001). Further regression analysis of S-PMA production rate on GSTT1 genotype with adjustment of sex, age, benzene exposure, and cotinine levels indicated that the genotype of GSTT1 plays a critical role in determining the inter-individual variations of S-PMA formation from benzene exposure. Therefore, the individual genotype of GSTT1 needs to be identified and considered while using S-PMA as a marker to estimate the personal exposure levels of benzene in future population studies.

We have previously reported that benzene decreases peripheral white blood cell and platelet counts and specifically lowers subsets of several blood cell types, including CD4+-T cells, B cells, NK cells, and granulocytes. Diminished thymus function has been implicated as a mechanism for CD4+-T cell loss in other conditions such as AIDS by assays of T cell receptor excision circles (TRECs), a marker of naive T cells that have recently emigrated from the thymus. To evaluate alteration of thymic function as a mechanism for benzene's effects on CD4+-T cell counts, we measured total TREC levels in 45 benzene-exposed workers and 45 unexposed controls. There was no significant difference in TREC levels per 10(6) peripheral blood leukocytes in the benzene-exposed workers compared to the controls. Although our study does not rule out counterbalancing alterations of TREC levels in specific T cell subsets, benzene's lymphotoxicity does not appear to be mediated through diminished thymus function.

We used cysteinyl adducts of serum albumin (Alb) to investigate the production of two reactive benzene metabolites, namely, benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) in workers exposed to benzene. Adducts were measured in 160 benzene-exposed workers who did not use respiratory protection (based upon individual geometric mean benzene exposure levels: median=5.27 ppm, interquartile range=2.14-13.4 ppm, range=0.074-328 ppm) and 101 local controls, from populations in Shanghai and Tianjin, China. After isolation of Alb, these adducts (designated as BO-Alb and 1,4-BQ-Alb) were cleaved from the protein with methanesulfonic acid and trifluoroacetic anhydride and measured by gas chromatography-mass spectrometry. Although BO-Alb and 1,4-BQ-Alb were measured in all subjects, levels of both adducts were 2.4-fold greater (median value) in exposed subjects than in controls (interquartile-fold range=1.63-4.05 for BO-Alb and 1.64-3.69 for 1,4-BQ-Alb). Log-log plots of the individual adduct levels versus exposure were quasi-linear with straight-line slopes of about 0.3 for both BO-Alb and 1,4-BQ-Alb. Since these log-space slopes were significantly less than one, we infer that adduct production was nonlinear, i.e., less-than proportional to benzene exposure, over the indicated range. This behavior points to saturation of CYP2E1 as a critical metabolic consequence of high exposure to benzene in humans. Thus, the biologically effective dose of BO and 1,4-BQ should be proportionally greater in persons exposed to low rather than high levels of benzene.

Benzene is known to have toxic effects on the blood and bone marrow, but its impact at levels below the U.S. occupational standard of 1 part per million (ppm) remains uncertain. In a study of 250 workers exposed to benzene, white blood cell and platelet counts were significantly lower than in 140 controls, even for exposure below 1 ppm in air. Progenitor cell colony formation significantly declined with increasing benzene exposure and was more sensitive to the effects of benzene than was the number of mature blood cells. Two genetic variants in key metabolizing enzymes, myeloperoxidase and NAD(P)H:quinone oxidoreductase, influenced susceptibility to benzene hematotoxicity. Thus, hematotoxicity from exposure to benzene occurred at air levels of 1 ppm or less and may be particularly evident among genetically susceptible subpopulations.

A gas chromatography-mass spectrometry method for measurement of the main urinary metabolites of benzene, namely, phenol, catechol, hydroquinone, 1,2,4-trihydroxybenzene (trihydroxybenzene), t,t-muconic acid (muconic acid), and S-phenylmercapturic acid (phenylmercapturic acid), is reported. The method is considerably simpler than existing assays. It was applied to urine from benzene-exposed subjects and controls from Shanghai, China. When subjects were divided into controls (n = 44), those exposed to 31 ppm benzene (n = 19), Spearman correlations with exposure category were >/= 0.728 (p < 0.0001) for all metabolites except trihydroxybenzene. When exposed subjects were compared on an individual basis, all metabolites, including trihydroxybenzene, were significantly correlated with benzene exposure (Pearson r >/= 0.472, p /= 0.708, p < 0.0001). Ratios of individual metabolite levels to total metabolite levels provided evidence of competitive inhibition of CYP 2E1 enzymes leading to increased production of phenol, catechol, and phenylmercapturic acid at the expense of hydroquinone, trihydroxybenzene, and muconic acid. Since all metabolites were detected in all control subjects, the method can be applied to persons exposed to environmental levels of benzene.

1,3-Butadiene (BD) is an important industrial chemical and pollutant. Its ability to induce genetic damage and cause hematological malignancies in humans is controversial. We have examined chromosome damage by fluorescence in situ hybridization (FISH) and mutations in the HPRT gene in the blood of Chinese workers exposed to BD. Peripheral blood samples were collected and cultured from 39 workers exposed to BD (median level 2 ppm, 6 h time-weighted average) and 38 matched controls in Yanshan, China. No difference in the level of aneuploidy or structural changes in chromosomes 1, 7, 8, and 12 was detected in metaphase cells from exposed subjects in comparison with matched controls, nor was there an increase in the frequency of HPRT mutations in the BD-exposed workers. Because genetic polymorphisms in glutathione S-transferase (GST) enzymes and microsomal epoxide hydrolase (EPHX1) may affect the genotoxic effects of BD and its metabolites, we also related chromosome alterations and gene mutations to GSTT1, GSTM1 and EPHX1 genotypes. Overall, there was no effect of variants in these genotypes on numerical or structural changes in chromosomes 1, 7, 8 and 12 or on HPRT mutant frequency in relation to BD exposure, but the GST genotypes did influence background levels of both hyperdiploidy and HPRT mutant frequency. In conclusion, our data show no increase in chromosomal aberrations or HPRT mutations among workers exposed to BD, even in potentially susceptible genetic subgroups. The study is, however, quite small and the levels of BD exposure are not extremely high, but our findings in China do support those from a similar study conducted in the Czech Republic. Together, these studies suggest that low levels of occupational BD exposure do not pose a significant risk of genetic damage.

Objectives: We carried out a detailed exposure assessment of benzene and toluene in two shoe factories in Tianjin, China. Our goal was to identify workers with a broad range of benzene exposures, for an epidemiologic study relating exposure to early biologic effects., Methods: A comprehensive exposure survey program was initiated. Over a period of 16 months, 2783 personal solvent exposure samples were collected in two workplaces from 250 workers. Mixed-effects models were used to identify factors affecting exposure. Principal component analyses (PCA) and subsequent regression analyses on the scores of the identified principal components were used to relate potential co-exposures to various exposure sources present in the workplace., Results: The mean benzene exposure level was 21.86 p.p.m. (10th-90th percentiles 5.23-50.63 p.p.m.) in the smaller shoe factory (factory A) and 3.46 p.p.m. (10th-90th percentiles 0.20-7.00 p.p.m.) in the larger shoe factory (factory B). Within-factory exposure levels differed among job titles and were higher for subjects directly involved in handling glues. In contrast, mean toluene levels were relatively similar in the two factories (factory A, 9.52 p.p.m.; factory B, 15.88 p.p.m.). A seasonal trend was identified for both benzene and toluene in factory B. This could be explained in part by changes in air movement and ventilation patterns occurring during the year. A seasonal trend was not present in the smaller shoe factory, where general ventilation was absent. Supplemental analysis showed that exposure levels to other hydrocarbons were low (< or =5 p.p.m.), less than 5% of their respective ACGIH threshold limit values, and generally comparable in the two factories. PCA showed that co-exposures in factory B could largely be explained by glue sources that were used in distinct areas in the workplace., Conclusions: We demonstrated the occurrence of a broad range of benzene exposure levels in two shoe manufacturing factories in Tianjin, China. Benzene and toluene exposures were determined in part by the degree of contact with glues, the benzene and toluene content of each glue, air movement and ventilation patterns. The availability of long-term monthly personal monitoring data provides an excellent opportunity to estimate individual exposures at different times during the 1 yr period of observation.

This article reports an extensive program to monitor individual personal exposures of subjects recruited for a study conducted in a Chinese occupational population to determine whether selected biological markers of exposure to benzene are reliable and sensitive enough to detect low-level benzene exposure in people. The monitoring program reported here was to assure an appropriate range of exposure for subject selection as well as to provide data for the exposure response assessment. The overall study resulted in correlation of the measured exposures with the measured concentrations of two minor urinary benzene metabolites, trans,trans-muconic acid and S-phenylmercapturic acid. The study design and evaluation of biological end points are presented in separate publications. Recruitment of 130 exposed subjects was based on personal exposure measurements collected with passive organic vapor monitors at weekly intervals for 3 to 4 weeks prior to collection of biological samples. Two monitors, side by side, were used for all of the personal monitoring in the first year of the study and about 10 percent of subsequent monitoring. One of each pair was analyzed immediately in Beijing at the Institute of Occupational Medicine, and the other was shipped to the United States and analyzed at the New York University Institute of Environmental Medicine. Exposure concentrations measured over 4-5 weeks were reasonably stable with average coefficients of variation of 0.58, 0.59, and 0.46 for benzene, toluene, and xylene, respectively. Benzene exposure averaged 10 +/- 13 ppm benzene with a median of 3.8 ppm for the recruited exposed workers. Excellent correlation was obtained between samples analyzed for benzene at the two laboratories. The extensive effort to document exposures was important to the exposure-response relationship demonstrated in the full study, which concluded that S-phenylmercapturic acid appears to be a good biomarker for detecting and evaluating benzene exposure at concentrations less than 0.25 ppm.

This study was conducted to validate biomarkers for early detection of benzene exposure and effect in 2 phases. The main purpose of phase 1 was to determine whether these biomarkers could reliably detect differences between workers with high exposure levels and unexposed subjects, which is the minimal screening criterion for a biomarker assay. Phase 2 of the study mainly focused on evaluating the exposure-response relation, confounding factors, and sensitivities of biomarkers for low benzene exposures. The Chinese occupational population studied had a broad range of benzene exposures. On the day of biological sample collection, exposures ranged from 0.06 to 122 ppm with a median exposure of 3.2 ppm. The median of the 4-week mean benzene exposures was 3.8 ppm, and the median lifetime cumulative exposure was 51.1 ppm-years. Compared with benzene levels in collected samples, toluene levels were relatively high, with a median of 12.6 ppm (mean, 26.3 ppm), but xylene levels were low, with a median of 0.30 ppm (mean, 0.40 ppm). The biomarkers evaluated were urinary metabolites S-phenylmercapturic acid (S-PMA*), trans,trans-muconic acid (t,t-MA), hydroquinone (HQ), catechol (CAT), and phenol; albumin adducts of benzene oxide and 1,4-benzoquinone (BO-Alb and 1,4-BQ-Alb, respectively) in blood; blood cell counts; and chromosomal aberrations. Blood cell counts in this population, including red blood cells (RBCs), white blood cells (WBCs), and neutrophils, decreased significantly with increased exposures but remained in normal ranges. Chromosomal aberration data showed significant increases of chromatid breaks and total chromosomal aberrations in exposed subjects compared with unexposed subjects. Among the urinary metabolites, the levels of S-PMA and t,t-MA were significantly elevated after benzene exposures. Both markers showed significant exposure-response trends even over the exposure range from 0 to 1 ppm. However, HQ, CAT, and phenol showed significant increases only for benzene exposure levels above 5 ppm. Multiple regression analyses of these urinary metabolites on benzene exposure indicated that toluene exposure, smoking status, and cotinine levels had no significant effects on urinary metabolite levels. A time-course study estimated the half-lives of S-PMA, t,t-MA, HQ, CAT, and phenol to be 12.8, 13.7, 12.7, 15.0, and 16.3 hours, respectively. Both BO-Alb and 1,4-BQ-Alb showed strong exposure-response associations with benzene. Regression analyses showed that after adjustment for potential confounding by smoking, there was still a strong association between benzene exposure and these markers. Furthermore, the analyses for correlations among biomarkers revealed that the urinary metabolites correlated substantially with each other. The albumin adducts also correlated well with the urinary biomarkers, especially with S-PMA. BO-Alb and 1,4-BQ adducts also correlated well with each other (r = 0.74). For benzene exposure monitoring, both S-PMA and t,t-MA were judged to be good and sensitive markers, which detected benzene exposures at around 0.1 ppm and 1 ppm, respectively. But S-PMA was clearly superior to t,t-MA as a biomarker for low levels of benzene exposure.

This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe-making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography-tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure., (Copyright 2002 Elsevier Science B.V.)

Background: Depression of peripheral blood cells is a well-known indicator of benzene hematotoxicity. Previous studies of its effects on specific types of blood cells have yielded inconsistent results. We examine hematological findings and their possible relations with exposure markers validated in a recent biomarker project conducted in Tianjin, China., Methods: Personal benzene exposures were sampled with 3-M organic vapor monitors, and analyzed by gas chromatography. The peripheral blood cells were counted by a cell counter. The WBC differential was manually counted on a total of 900 cells by a US commercial laboratory., Results: A total of 130 exposed workers and 51 age- and gender-matched unexposed subjects were recruited in this study. Benzene exposure levels monitored on the day of biological sample collection for exposed workers ranged from 0.06 to 122 ppm. Their 4-week average and cumulative benzene exposure levels were 0.08-54.5 ppm and 6.1-623.2 ppm-years, respectively. Significant decreases of red blood cells (RBC), white blood cells (WBC), and neutrophils were observed and correlated with both personal benzene exposures and levels of urinary metabolites (S-phenylmercapuric acid and t,t-muconic acid) and albumin adducts of benzene oxide and 1,4-benzeoquinone., Conclusions: The depressions in RBC, WBC, and neutrophils observed in this study are not only exposure dependent, but also significantly different in the lowest exposed group (at or below 0.25 ppm) compared with unexposed subjects. The results of the present study appear to suggest that lymphocytes may not be more sensitive to chronic benzene exposure than neutrophils., (Copyright 2002 Wiley-Liss, Inc.)

Albumin adducts of benzene oxide (BO-Alb) and 1,4-benzoquinone (1,4-BQ-Alb) were investigated among 134 workers exposed to benzene and 51 unexposed controls in Tianjin, China. Concentrations of both adducts increased with benzene exposure [range = 0.07-46.6 parts/million (ppm); median = 3.55 ppm] and with urinary cotinine. Adduct levels were less than proportional to benzene exposure, suggesting saturable CYP 2E1 metabolism of benzene. Because the transition from linear to saturable metabolism began at approximately 1 ppm, the common assumption of linear kinetics at much higher benzene exposures could lead to substantial underestimation of leukemia risks. Adduct levels were generally lower in older workers, indicating that CYP 2E1 metabolism diminished with age, at approximately 2%/year of life. The ratio of 1,4-BQ-Alb:BO-Alb decreased with age and coexposure to toluene, and increased with alcohol consumption. This indicates that factors affecting CYP 2E1 metabolism exerted a greater role on production of 1,4-BQ than BO, presumably because of the second oxidation step from phenol to hydroquinone. The adduct ratio was also positively associated with urinary cotinine, suggesting that both benzene and hydroquinone from cigarette smoke affected adduct levels. Results of a limited time course study of 11 subjects indicated moderate chemical instability of 1,4-BQ-Alb (half life = 13.5 days compared with 21 days for normal Alb turnover), whereas no evidence of instability of BO-Alb was observed. This study illustrates that Alb adducts can be used to investigate the dispositions of reactive metabolites of procarcinogens in humans, provided that exposures are adequately characterized in the month preceding blood collection.

Background: Cancer screening with highly sensitive, specific biomarkers that reflect molecular phenotypic alterations is an attractive strategy for cancer control. We examined whether biomarker profiles could be used for risk assessment and cancer detection in a cohort of Chinese workers occupationally exposed to benzidine and at risk for bladder cancer., Methods: The cohort consisted of 1788 exposed and 373 nonexposed workers, followed from 1991 through 1997. We assayed urothelial cells from voided urine samples for DNA ploidy (expressed as the 5C-exceeding rate [DNA 5CER]), the bladder tumor-associated antigen p300, and a cytoskeletal protein (G-actin). Workers were stratified into different risk groups (high, moderate, and low risk) at each examination based on a predefined biomarker profile. For workers who developed bladder cancer, tumor risk assessment was analyzed from samples collected 6-12 months before the cancer diagnosis. The associations between risk group and subsequent development of bladder cancer were analyzed by Cox proportional hazards regression analysis and logistic analysis, after adjustment. All statistical tests were two-sided., Results: Twenty-eight bladder cancers were diagnosed in exposed workers and two in nonexposed workers. For risk assessment, DNA 5CER had 87.5% sensitivity, 86.5% specificity, an odds ratio (OR) of 46.2 (95% confidence interval [CI] = 8.1 to 867.0), and a risk ratio (RR) of 16.2 (95% CI = 7.1 to 37.0); p300 had 50.0% sensitivity, 97.9% specificity, an OR of 40.0 (95% CI = 9.0 to 177.8), and an RR of 37.9 (95% CI = 16.8 to 85.3). The risk of developing bladder cancer was 19.6 (95% CI = 8.0 to 47.9) times higher in workers positive for either the DNA 5CER or p300 biomarkers than in workers negative for both biomarkers and 81.4 (95% CI = 33.3 to 199.3) times higher in workers positive for both biomarkers. G-actin was a poor marker of individual risk., Conclusions: Occupationally exposed workers at risk for bladder cancer can be individually stratified, screened, monitored, and diagnosed based on predefined molecular biomarker profiles.

Urinary benzene (UB) was investigated as a biomarker of exposure among benzene-exposed workers and unexposed subjects in Shanghai, China. Measurements were performed via headspace solid phase microextraction of 0.5 ml of urine specimens followed by gas chromatography-mass spectrometry. This assay is simple and more sensitive than other methods (detection limit 0.016 microg benzene/l urine). The median daily benzene exposure was 31 p.p.m. (range 1.65-329 p.p.m.). When subjects were divided into controls (n = 41), those exposed to < or =31 p.p.m. benzene (n = 22) and >31 p.p.m. benzene (n = 20), the median UB levels were 0.069, 4.95 and 46.1 microg/l, respectively (Spearman r = 0.879, P < 0.0001). A linear relationship was observed between the logarithm of UB and the logarithm of benzene exposure in exposed subjects according to the following equation: ln(UB, microg/l) = 0.196 + 0.709 ln (exposure, p.p.m.) (r = 0.717, P < 0.0001). Considering all subjects, linear relationships were also observed between the logarithm of UB and the corresponding logarithms of four urinary metabolites of benzene, namely t,t-muconic acid (r = 0.938, P < 0.0001), phenol (r = 0.826, P < 0.0001), catechol (r = 0.812, P < 0.0001) and hydroquinone (r = 0.898, P: < 0.0001). Ratios of individual metabolite levels to total metabolites versus UB provide evidence of competitive inhibition of CYP450 enzymes leading to increased production of phenol and catechol at the expense of hydroquinone and muconic acid. Among control subjects UB was readily detected with a mean level of 0.145 microg/l (range 0.027-2.06 microg/l), compared with 5.63 microg/l (range 0.837-26.38 microg/l) in workers exposed to benzene below 10 p.p.m. (P < 0.0001). This suggests that UB is a good biomarker for exposure to low levels of benzene.

Background: The present study was conducted among Chinese workers employed in glue- and shoe-making factories who had an average daily personal benzene exposure of 31+/-26 ppm (mean+/-SD). The metabolites monitored were S-phenylmercapturic acid (S-PMA), trans, trans-muconic acid (t,t-MA), hydroquinone (HQ), catechol (CAT), 1,2, 4-trihydroxybenzene (benzene triol, BT), and phenol., Methods: S-PMA, t,t-MA, HQ, CAT, and BT were quantified by HPLC-tandem mass spectrometry. Phenol was measured by GC-MS., Results: Levels of benzene metabolites (except BT) measured in urine samples collected from exposed workers at the end of workshift were significantly higher than those measured in unexposed subjects (P < 0.0001). The large increases in urinary metabolites from before to after work strongly correlated with benzene exposure. Concentrations of these metabolites in urine samples collected from exposed workers before work were also significantly higher than those from unexposed subjects. The half-lives of S-PMA, t,t-MA, HQ, CAT, and phenol were estimated from a time course study to be 12.8, 13.7, 12.7, 15.0, and 16.3 h, respectively., Conclusions: All metabolites, except BT, are good markers for benzene exposure at the observed levels; however, due to their high background, HQ, CAT, and phenol may not distinguish unexposed subjects from workers exposed to benzene at low ambient levels. S-PMA and t,t-MA are the most sensitive markers for low level benzene exposure., (Copyright 2000 Wiley-Liss, Inc.)

While 1,3-butadiene is carcinogenic in rodents, cancer causation in humans is less certain. We examined a spectrum of genotoxic outcomes in 41 butadiene polymer production workers and 38 non-exposed controls, in China, to explore the role of butadiene in human carcinogenesis. Because in vitro studies suggest that genetic polymorphisms in glutathione S-transferase enzymes influence genotoxic effects of butadiene, we also related genotoxicity to genetic polymorphisms in GSTT1 and GSTM1. Among butadiene-exposed workers, median air exposure was 2 p.p.m. (6 h time-weighted average), due largely to intermittent high level exposures. Compared with unexposed subjects, butadiene-exposed workers had greater levels of hemoglobin N-(2,3,4-trihydroxybutyl)valine (THBVal) adducts (P < 0.0001) and adduct levels tended to correlate, among butadiene-exposed workers, with air measures (P = 0.03). Butadiene-exposed workers did not differ, however, from unexposed workers with respect to frequency of uninduced or diepoxybutane-induced sister chromatid exchanges, aneuploidy as measured by fluorescence in situ hybridization of chromosomes 1, 7, 8 and 12, glycophorin A variants or lymphocyte hprt somatic mutation. Also among the exposed, greater THBVal levels were not associated with increases in uninduced sister chromatid exchanges, aneuploidy, glycophorin A or hprt mutations. Butadiene-exposed workers had greater lymphocyte (P = 0.002) and platelet counts (P = 0.07) and lymphocytes as a percentage of white blood cells were moderately correlated with greater THBVal levels (Spearman's phi = 0.32, P = 0.07). Among butadiene-exposed workers, neither GSTM1 nor GSTT1 genotype status predicted urinary mercapturic acid butanediol formation, THBVal adducts, uninduced sister chromatid exchanges, aneuploidy or mutations in the glycophorin A or hprt genes. Overall, the study demonstrated exposure to butadiene in these workers, by a variety of short-term and long-term measures, but did not show specific genotoxic effects, at the chromosomal or gene levels, related to that exposure.

This paper described the concept of moleculalr epidemiology, it's differences from traditional epidemiology and what the author learned from conducting molecular epidemiology recently. 1. Molecular epidemiology is the borderline subject in combination of molecular biology with epidemiology. 2. The level of chemical carcinogen formed in vivo and the amount of combination of chemical carcinogen either in the level from vivo or in biologic effective dose of combination of chemical carcinogen with macromolecular are all related to cancer development. 3. The syndrome of chromosome aberration is an early biologic effect in cancer process and oncogen activation or suppressing gene inactivation might be necessary for malignant transformation from initiated cell. 4. Individual susceptibility could be affected by host and acquired factors. 5. Biologic bank could provide continuous observation and comparision between before and after cancer formation.

Benzene is an established human leukemogen that increases the level of chromosome aberrations in lymphocytes of exposed workers. Numerical aberrations (aneusomy) can be observed by fluorescence in situ hybridization (FISH) in both interphase and metaphase cells. Whereas interphase FISH allows nondividing cells to be analyzed, one advantage of metaphase FISH is that it can also detect structural changes. The present study compares the abilities of metaphase and interphase FISH to detect aneusomy of chromosomes 7 and 8 in healthy benzene-exposed human subjects. Metaphase and interphase cells from the peripheral blood of 43 workers exposed to benzene (median = 31 ppm, 8-hr TWA) and 44 frequency-matched controls were analyzed by FISH. Normal diploid cells contained two hybridization signals, whereas those that were potentially monosomic contained one, trisomic 3 and tetrasomic 4. The frequency of cells with one hybridization signal for chromosome 7 in metaphase spreads rose from 2.72 +/- 0.19 (%, mean +/- SE) in controls to 3.79 +/- 0.63 in workers exposed to 31 or fewer ppm benzene and 5.9 +/- 0.85 in those exposed to more than 31 ppm (P(trend) < 0.0001). No similar dose-dependent increase in the frequency of cells with one hybridization signal was observed by interphase FISH, probably because of probe overlap artifact. Although significant dose-dependent increases in the frequency of cells with three hybridization signals for chromosome 7 were detected by both methods in the higher-exposed group, a larger, more significant difference was detected by metaphase FISH between controls and workers exposed to 31 or fewer ppm. Similar data were obtained for chromosome 8. Interphase and metaphase FISH were moderately correlated for three hybridization signals but not for one hybridization signal in chromosome 7 or 8. In general, metaphase FISH was more sensitive in detecting both monosomy and trisomy in the lymphocytes of exposed workers., (Copyright 1999 Wiley-Liss, Inc.)

Two of the most common cytogenetic changes in therapy- and chemical-related leukemia are the loss and long (q) arm deletion of chromosomes 5 and 7. The detection of these aberrations in lymphocytes of individuals exposed to potential leukemogens may serve as useful biomarkers of increased leukemia risk. We have used a novel fluorescence in situ hybridization (FISH) procedure to determine if specific aberrations in chromosomes 1, 5 and 7 occur at an elevated rate in the blood cells of workers exposed to benzene. Forty-three healthy workers exposed to a wide range of benzene concentrations (median 31 p.p.m., 8 h time-weighted average) and 44 unexposed controls from Shanghai were studied. Whole blood was cultured and metaphase spreads were harvested at 72 h. Benzene exposure was associated with increases in the rates of monosomy 5 and 7 but not monosomy 1 (P < 0.001, P < 0.0001 and P = 0.94, respectively) and with increases in trisomy and tetrasomy frequencies of all three chromosomes. Long arm deletion of chromosomes 5 and 7 was increased in a dose-dependent fashion (P = 0.014 and P < 0.0001) up to 3.5-fold in the exposed workers. These results demonstrate that leukemia-specific changes in chromosomes 5 and 7 can be detected by FISH in the peripheral blood of otherwise healthy benzene-exposed workers. We suggest that aberrations in chromosomes 5 and 7 may be useful biomarkers of early biological effect for benzene exposure.

Objectives: Animal inhalation studies and theoretical models suggest that the pattern of formation of benzene metabolites changes as exposure to benzene increases. To determine if this occurs in humans, benzene metabolites in urine samples collected as part of a cross sectional study of occupationally exposed workers in Shanghai, China were measured., Methods: With organic vapour monitoring badges, 38 subjects were monitored during their full workshift for inhalation exposure to benzene. The benzene urinary metabolites phenol, catechol, hydroquinone, and muconic acid were measured with an isotope dilution gas chromatography mass spectroscopy assay and strongly correlated with concentrations of benzene air. For the subgroup of workers (n = 27) with urinary phenol > 50 ng/g creatinine (above which phenol is considered to be a specific indicator of exposure to benzene), concentrations of each of the four metabolites were calculated as a ratio of the sum of the concentrations of all four metabolites (total metabolites) and were compared in workers exposed to > 25 ppm v < or = 25 ppm., Results: The median, 8 hour time weighted average exposure to benzene was 25 ppm. Relative to the lower exposed workers, the ratio of phenol and catechol to total metabolites increased by 6.0% (p = 0.04) and 22.2% (p = 0.007), respectively, in the more highly exposed workers. By contrast, the ratio of hydroquinone and muconic acid to total metabolites decreased by 18.8% (p = 0.04) and 26.7% (p = 0.006), respectively. Similar patterns were found when metabolite ratios were analysed as a function of internal benzene dose (defined as total urinary benzene metabolites), although catechol showed a more complex, quadratic relation with increasing dose., Conclusions: These results, which are consistent with previous animal studies, show that the relative production of benzene metabolites is a function of exposure level. If the toxic benzene metabolites are assumed to be derived from hydroquinone, ring opened products, or both, these results suggests that the risk for adverse health outcomes due to exposure to benzene may have a supralinear relation with external dose, and that linear extrapolation of the toxic effects of benzene in highly exposed workers to lower levels of exposure may underestimate risk.

Benzene oxide (BO) reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts (BO-Hb and BO-Alb), which are presumed to be specific biomarkers of exposure to benzene. We analyzed BO-Hb in 43 exposed workers and 42 unexposed controls, and BO-Alb in a subsample consisting of 19 workers and 19 controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. The adducts were analyzed by gas chromatography-mass spectrometry following reaction of the protein with trifluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to < or =31 (n = 21) and >31 p.p.m. (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol/g globin, respectively (correlation with exposure: Spearman r = 0.67, P < 0.0001). To our knowledge, these results represent the first observation in humans that BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these 3 groups were 103 (n = 19), 351 (n = 7) and 2010 (n = 12) pmol/g Alb, respectively, also reflecting a significant correlation with exposure (Spearman r = 0.90, P < 0.0001). The blood dose of BO predicted from both Hb and Alb adducts was very similar. These results clearly affirm the use of both Hb and Alb adducts of BO as biomarkers of exposure to high levels of benzene. As part of our investigation of the background levels of BO-Hb and BO-Alb found in unexposed persons, we analyzed recombinant human Hb and Alb for BO adducts. Significant levels of both BO-Hb (19.7 pmol/g) and BO-Alb (41.9 pmol/g) were detected, suggesting that portions of the observed background adducts reflect an artifact of the assay, while other portions are indicative of either unknown exposures or endogenous production of adducts.

Chromosome aberrations in peripheral blood lymphocytes have been used for many years to monitor human populations exposed to potential carcinogens. Recent reports have confirmed the validity of this approach by demonstrating that elevated levels of chromosome aberrations in lymphocytes are associated with subsequent increased cancer risk, especially for increased mortality from hematological malignancies including acute myeloid leukemia (AML). We postulated that this approach could be improved in two ways: (a) by detecting oncogenic disease-specific aberrations; and (b) by using chromosome painting so that many more metaphases could be analyzed. Numerical and structural aberrations in chromosomes 8 and 21 are commonly observed in AML. In the present study, we painted chromosomes 8 and 21 in lymphocyte metaphases from 43 healthy workers exposed to benzene, an established cause of AML, and from 44 matched controls. To examine dose-response relationships the workers were divided into two groups at the median exposure level, a lower-exposed group (< or = 31 ppm; n = 21), and a higher-exposed group (> 31 ppm; n = 22). Benzene exposure was associated with significant increases in hyperdiploidy of chromosomes 8 (1.2, 1.5, and 2.4 per 100 metaphases; P < 0.0001) and 21 (0.9, 1.1, and 1.9 per 100 metaphases; P < 0.0001). Translocations between chromosomes 8 and 21 were increased up to 15-fold in highly exposed workers (0.01, 0.04, and 0.16 per 100 metaphases; P < 0.0001). In one highly exposed individual, these translocations were reciprocal and were detectable by reverse transcriptase-PCR. These data indicate a potential role for t(8;21) in benzene-induced leukemogenesis and are consistent with the hypothesis that detection of specific chromosome aberrations may be a powerful approach to identify populations at increased risk of leukemia.

The frequency of micronuclei (MN) in cells of the nasal mucosa, oral mucosa and in lymphocytes was evaluated for 25 students in anatomy classes exposed to formaldehyde (FA) over an 8-week period. Each student served as his or her own control. The time-weighted average concentration (TWA) of formaldehyde in anatomical laboratories and in students' dormitories was 0.508 +/- 0.299 mg/m3 and 0.012 +/- 0.0025 mg/m3, respectively. A higher frequency of micronuclei was observed in nasal and oral exfoliative cells after formaldehyde exposure (3.85 +/- 1.48 vs 1.20 +/- 0.676 and 0.857 +/- 0.558 vs 0.568 +/- 0.317, paired-t test: P < 0.001 and P < 0.01, respectively). No significant increase in the frequency of lymphocyte micronuclei was found after formaldehyde exposure (P > 0.05). The present study shows that nasal mucosa cells exposed through respiration are the chief target of FA-induced genotoxic effects.

Background: Benzene is a widely distributed environmental contaminant known to cause leukemia, particularly acute nonlymphocytic leukemia, and perhaps other hematologic neoplasms and disorders. Few epidemiologic studies, however, have been able to address relationships between the extent of benzene exposure and the level of risk., Purpose: A large cohort study was carried out in China to evaluate the risks of developing specific hematologic neoplasms and selected related disorders in relationship to quantitative estimates of occupational benzene exposure., Methods: A cohort of 74828 benzene-exposed and 35805 unexposed workers employed from 1972 through 1987 in 12 cities in China was identified and followed to determine the incidence of hematologic neoplasms and related disorders. Estimates of benzene exposure were derived from work histories and available historic benzene measurements. Existing pathologic material and supporting medical records were reviewed to establish diagnoses of disease. Relative risks (RRs) (i.e., ratios of incidence rates for specific hematologic neoplasms and related disorders in the benzene-exposed group to incidence rates in the unexposed group) were determined by use of Poisson regression analysis, with stratification by age and sex., Results: For workers historically exposed to benzene at average levels of less than 10 parts per million (ppm), the RR for all hematologic neoplasm combined was 2.2 (95% confidence interval [CI] = 1.1-4.2), and, for the combination of acute nonlymphocytic leukemia and related myelodysplastic syndromes, the RR was 3.2 (95% CI = 1.0-10.1). For individuals who were occupationally exposed to benzene at constant levels of 25 ppm or more, the RR for the combination of acute nonlymphocytic leukemia and related myelodysplastic syndromes was 7.1 (95% CI = 2.1-23.7). Workers with 10 or more years of benzene exposure had an RR of developing non-Hodgkin's lymphoma of 4.2 (95% CI = 1.1-15.9), and the development of this neoplasm was linked most strongly to exposure that had occurred at least 10 years before diagnosis (i.e., distant exposure) (P for trend = .005, two-sided). In contrast, the risk for the combination of acute nonlymphocytic leukemia and related myelodysplastic syndromes was significantly increased among those with more recent benzene exposure (P for trend = .003, two-sided), but it was not linked to distant exposure (P for trend = .51, two-sided)., Conclusions: The results of this study suggest that benzene exposure is associated with a spectrum of hematologic neoplasms and related disorders in humans. Risks for these conditions are elevated at average benzene-exposure levels of less than 10 ppm and show a tendency, although not a strong one, to rise with increasing levels of exposure. The temporal pattern of benzene exposure appears to be important in determining the risk of developing specific diseases.

Benzene is a ubiquitous occupational hematotoxin and leukemogen, but people vary in their response to this toxic agent. To evaluate the impact of interindividual variation in enzymes that activate (i.e., CYP2E1) and detoxify (i.e., NQO1) benzene and its metabolites, we carried out a case-control study in Shanghai, China, of occupational benzene poisoning (BP; i.e., hematotoxicity), which we show is itself strongly associated with subsequent development of acute nonlymphocytic leukemia and the related myelodysplastic syndromes (relative risk, 70.6; 95% confidence interval, 11.4-439.3). CYP2E1 and NQO1 genotypes were determined by PCR-RFLP, and CYP2E1 enzymatic activity was estimated by the fractional excretion of chlorzoxazone (fe(6-OH)) for 50 cases of BP and 50 controls. Subjects with both a rapid fe(6-OH). and two copies of the NQO1 609C-->T mutation had a 7.6-fold (95% confidence interval, 1.8-31.2) increased risk of BP compared to subjects with a slow fe(6-OH) who carried one or two wild-type NQO1 alleles. In contrast, the CYP2E1 PstI/RsaI polymorphism did not influence BP risk. This is the first report that provides evidence of human susceptibility to benzene-related disease. Further evaluation of susceptibility for hematotoxicity and hematological malignancy among workers with a history of occupational exposure to benzene is warranted.

A large cohort of 74,828 benzene-exposed and 35,805 nonexposed workers employed between 1972 and 1987 in 12 cities in China was followed to determine mortality from all causes. Benzene-exposed study subjects were employed in a variety of occupations including coating applications, and rubber, chemical, and shoe production. Mortality was slightly increased among workers with greater cumulative exposure to benzene (ptrend < 0.05), but this excess was largely due to cancer deaths (ptrend < 0.01). Deaths due to lymphatic and hematopoietic malignancies (ptrend = 0.01) and lung cancer (ptrend = 0.01) increased with increasing cumulative exposure to benzene. Investigations continue to relate benzene exposure to specific lymphatic and hematopoietic malignancies and other causes of death.

Benzene is a recognized hematotoxin and leukemogen, but its mechanisms of action in humans are still uncertain. To provide insight into these processes, we carried out a cross-sectional study of 44 healthy workers currently exposed to benzene (median 8-hr time-weighted average; 31 ppm), and unexposed controls in Shanghai, China. Here we provide an overview of the study results on peripheral blood cells levels and somatic cell mutation frequency measured by the glycophorin A (GPA) gene loss assay and report on peripheral cytokine levels. All peripheral blood cells levels (i.e., total white blood cells, absolute lymphocyte count, platelets, red blood cells, and hemoglobin) were decreased among exposed workers compared to controls, with the exception of the red blood cell mean corpuscular volume, which was higher among exposed subjects. In contrast, peripheral cytokine levels (interleukin-3, interleukin-6, erythropoietin, granulocyte colony-stimulating factor, tissue necrosis factor-alpha) in a subset of the most highly exposed workers (n = 11) were similar to values in controls (n = 11), suggesting that benzene does not affect these growth factor levels in peripheral blood. The GPA assay measures stem cell or precursor erythroid cell mutations expressed in peripheral red blood cells of MN heterozygous subjects, identifying NN variants, which result from loss of the GPA M allele and duplication of the N allele, and N phi variants, which arise from gene inactivation. The NN (but not N phi) GPA variant cell frequency was elevated in the exposed workers compared with controls (mean +/- SD, 13.9 +/- 8.4 mutants per million cells versus 7.4 +/- 5.2 per million cells, (respectively; p = 0.0002), suggesting that benzene produces gene-duplicating but not gene-inactivating mutations at the GPA locus in bone marrow cells of exposed humans. These findings, combined with ongoing analyses of benzene macromolecular adducts and chromosomal aberrations, will provide an opportunity to comprehensively evaluate a wide range of early biologic effects associated with benzene exposure in humans.

An expanded cohort study of 74,828 benzene-exposed and 35,805 unexposed workers were followed during 1972 to 1987, based on a previous study in 12 cities in China. A small increase was observed in total cancer mortality among benzene-exposed compared with unexposed workers (relative risk [RR] = 1.2). Statistically significant excesses were noted for leukemia (RR = 2.3), malignant lymphoma (RR = 4.5), and lung cancer (RR = 1.4). When risks were evaluated by leukemia subtype, only acute myelogenous leukemia was significantly elevated (RR = 3.1), although nonsignificant excesses were also noted for chronic myelogenous leukemia (RR = 2.6) and acute lymphocytic leukemia (RR = 2.3). A significant excess was also found for aplastic anemia.