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 cell levels and somatic cell mutation frequency measured by the glycophorin A (GPA) gene loss assay and report on peripheral cytokine levels. All peripheral blood cell 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 colonystimulating factor, tissue necrosis factor-a) 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 No variants, which arise from gene inactivation. The NN (but not NO) 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.

Occupational exposure to benzene is known to cause leukemia, but the mechanism remains unclear. Unlike most other carcinogens, benzene and its metabolites are weakly or nonmutagenic in most simple gene mutation assays. Benzene and its metabolites do, however, produce chromosomal damage in a variety of systems. Here, we have used the glycophorin A (GPA) gene loss mutation assay to evaluate the nature of DNA damage produced by benzene in 24 workers heavily exposed to benzene and 23 matched control individuals in Shanghai, China. The GPA assay identifies stem cell or precursor erythroid cell mutations expressed in peripheral erythrocytes of MN-heterozygous subjects, distinguishing the NN and N0 mutant variants. A significant increase in the NN GPA variant cell frequency (Vf) was found in benzene-exposed workers as compared with unexposed control individuals (mean ± SEM, 13.9 ± 1.7 per million cells vs. 7.4 + 1.1 per million cells in control individuals; P = 0.0002). In contrast, no significant difference existed between the two groups for the N0 Vf (9.1 + 0.9 vs. 8.8 + 1.8 per million cells; P = 0.21). Further, lifetime cumulative occupational exposure to benzene was associated with the NN Vf (P = 0.005) but not with the N0 Vf (P = 0.31), suggesting that NN mutations occur in longer-lived bone marrow stem cells. NN variants result from loss of the GPA M allele and duplication of the N allele, presumably through recombination mechanisms, whereas N0 variants arise from gene inactivation, presumably due to point mutations and deletions. Thus, these results suggest that benzene produces gene-duplicating mutations but does not produce gene-inactivating mutations at the GPA locus in bone marrow cells of humans exposed to high benzene levels. This finding is consistent with data on the genetic toxicology of benzene and its metabolites and adds further weight to the hypothesis that chromosome damage and mitotic recombination are important in benzene-induced leukemia.

Previous investigations suggest that dietary sources of polycyclic aromatic hydrocarbons (PAHs) contribute to the PAH-DNA adduct load in peripheral white blood cells (WBCs). In the current study, we measured PAH-DNA adducts by enzyme-linked immunosorbent assay in WBCs obtained from 47 California wildland (forest) firefighters at two time points (early and late) during an active forest fire season. PAH-DNA adduct levels were not associated with recent firefighting activity, but were positively associated with frequency of charbroiled food consumption in the previous 2 weeks. In addition, adduct levels declined with time since last ingestion of charbroiled food. These studies indicate that recent consumption of charbroiled food contributes to the PAH-DNA adduct load in peripheral WBCs.

A rat genomic DNA fragment containing a tRNA gene cluster was isolated from a lambda phage library. HybridizatUa and nucleotide sequenie analysis revealed the presence of a 83 bp tRNAC gene and a 72 bp tRNA P gene. Both genes possessed intact coding regioCii and putative trans-GUG cription termination signals at their respective 3' ends. In vitro transcription analysis of the two subcloned genes in a HeLa cell S-100 system demonstrated the specific synthesis of a number of RNAs by RNA polymerase III. Studies carried out in the presence of a-amanitin showed that the larger RNAs are precursors for the final processed transcripts of the tRNALeu and tRNA P genes, respectively. Further pucleotide se8 ence analysis of the cluster revealed the presence of tRNA Y and a tRNA pseudogenes with missing areas within their coding regions which are essential for transcription by RIA polymerase III. Within the region of DNA between the tRNA Le and tRNA P genes is a sequence which is 65% homologous to a region of the rat Bi element. The significance of this latter structure within the gene cluster is unknown.