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Investigation of benzene-DNA adducts and their detection in human bone marrow.

We have examined DNA adduct formation in HL-60 cells and human bone marrow treated with either hydroquinone or p-benzoquinone and have found that these treatments produce the same DNA adduct in both cell types. The DNA adduct level from these treatments varied from 0.05 to 7.5 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. Reaction of calf thymus DNA with p-benzoquinone produced three adducts as detected by 32P-postlabeling. These adducts have been identified as (3'-hydroxy)-3,N4-benzetheno-2'-deoxycytidine-3'-phosphate; (3'-hydroxy)-1,N6-benzetheno-2'-deoxyadenosine-3'-phosphate; and (3'-hydroxy)-1,N2-benzetheno-2'-deoxyguanosine-3'-phosphate. The DNA adduct formed in HL-60 cells did not correspond to any of the principal adducts formed in DNA reacted with p-benzoquinone, suggesting that cellular environment modifies DNA adduct production by p-benzoquinone. These studies demonstrate that DNA adduct formation occurs in human bone marrow treated with benzene metabolites and suggest that P1-enhanced 32P-postlabeling may be used to detect DNA adducts resulting from benzene exposure.


Introduction
Human exposure to benzene can occur from both occupational andenvironmental sources (1). Inaddition, aprincipal sourceof benzene exposure is cigarette smoke (2). Benzene is carcinogenic in rats and mice (3) and leukemogenic in highly exposed humans (4). Recent epidemiologlc evidence also suggests that smoking is associated with an increased risk for nonlymphocytic leukemia (5). These results raise the question as to the risk associated with benzene-exposure and if measurement ofbenzene-DNA adducts can be used as a dosimeter of exposure.
Initial benzene metabolism occurs in the liver with formation of benzene oxide by P-450 oxidation followed by spontaneous conversion to phenol. This primary metabolite is further oxidized to hydroquinone (HQ) and catechol (6). These metabolites have been shown to accumulate in the bone marrow (7), and it has been postulated that myeloperoxidases (MPO) present in the myeloid bone marrow cells may convert HQ top-benzoquinone (pBQ) (8).
The PI-nuclease-enhanced "P-postlabeling procedure has been shown to be a very sensitive method for the detection and quantitation of aromatic DNA adducts (9). In the present study we have used this procedure to measure DNA adduct formation in HL-60 cells and human bone marrow treated with the benzene metabolitespBQ and HQ.

Detection of DNA Adducts in Human Bone Marrow
Bone marrow is the target tissue for leukemogenic effects benzene in humans. For our study we have obtained bone marrow samples from patients undergoingjoint replacement operations. Treatment of bone marrow with 250 1.M ofpBQ for 2 hr produced a single DNA adduct in bone marrow cells (Fig. 4A). Co-chromatography studies of 32P-postlabeled DNA from bone show that the adduct formed in bone marrow cells is identical to that formed in HL-60 cells. (Fig. 4B).
Comparison ofthe extent of DNA adduct formation in HL-60 cells and bone marrow after HQ andpBQ treatment was made by analyzing the slopes ofthe dose-response curves (Fig. 5). For HQ treatment the slopes were 19.8 x 10-4 for HL-60 and 5.16 x 10-4 for bone marrow, where as forpBQ treatment the slopes were 21.6 x 1i-3 and 3.14 x 1O-3 for HL-60 and bone marrow, respectively. Comparison of these values shows that HQ is approximately 4to 5-fold andpBQ is approximately 7to 8-fold more effective at adduct formation in HL-60 cells compared to bone marrow. A possible explanation for the reduced binding in bone marrow is that this tissue contains both erythroid and myeloid cells and only the myeloid cells have the capacity to activate HQ. These studies demonstrate that HL-60 cells are a good model for the activation and binding of benzene metabolites in the myeloid cells of human bone marrow.

Comparison of Adducts Formed in DNA and in Cells
DNA isolated from HL-60 cells treated with 250 1iM of HQ for 8 hr was 32P-postlabeled and co-chromatographed with 32p_ postlabeled DNA reacted with pBQ. Figure 6 shows that the cellular adduct as indicated did not correspond to any ofthe major adducts formed in DNA.

Conclusion
The DNA adducts formed in HL-60 cells and bone marrow treated either withpBQ or with HQ are identical. These results are consistent with the requirement for HQ to be enzymatically oxidized topBQ to form DNA adducts. The mechanism for activation of HQ is probably via MPO (8). The DNA adduct formed in HL-60 cells treated with either pBQ or HQ did not  Our studies have identified a DNA adduct formed by the benzene metabolites hydroquinone andpBQ in human bone marrow and suggest that DNA adduct formation by these metabolites may be involved in benzene-induced leukemogenesis. The application of the P1-enhanced 32P-postlabeling procedure should allow for detection ofbenzene-DNA adducts in populations exposed to benzene either occupationally or from tobacco smoke. However, these results also indicate that cells containing peroxidase activity such as neutrophils would be the appropriate cell type for monitoring the effects of benzene exposure, rather than lymphocytes, which do not contain peroxidase activity.