Alan M. Jeffrey

Biologic markers in ethylene oxide-exposed workers and controls

Ethylene oxide (EtO) is an alkylating agent and a model direct-acting mutagen and carcinogen. This study has evaluated a panel of biologic markers including EtO-hemoglobin adducts (EtO-Hb), sister-chromatid exchanges (SCEs), micronuclei, chromosomal aberrations (CAs), DNA single-strand breaks (SSB) and an index of DNA repair (ratio of UDS to NA-AAF-DNA binding) in the peripheral blood cells of 34 workers at a sterilization unit of a large university hospital and 23 controls working in the university library. Comprehensive environmental histories were obtained on each subject including detailed occupational and smoking histories. Industrial hygiene data obtained prior to the study and personal monitoring during the 8 years preceding the study showed that workers were subject to low-level exposure near or below the current Occupational Safety and Health Administration (OSHA) standard of 1 ppm (TWA). Personal monitoring data obtained during 2 weeks prior to blood sampling were uniformly less than 0.3 ppm (TWA). After adjusting for smoking, EtO workplace exposure was significantly (p less than 0.001) associated with EtO-Hb (a carcinogen-protein adduct) and 2 measures of SCEs [the average number of SCEs/cell (SCE50) and the number of high frequency cells (SCEHFC)]. There was an apparent suppression of DNA repair capacity in EtO-exposed individuals as measured by the DNA repair index; i.e., the ratio of unscheduled DNA synthesis (UDS) and NA-AAF-DNA binding (p less than 0.01). No association of DNA repair index with smoking was found. Another important finding of this study is the highly significant correlation between EtO-Hb adduct levels and SCEHFC (p less than 0.01) and SCEs (p less than 0.02) which provides evidence of a direct link between a marker of biologically effective dose and markers of genotoxic response. In contrast, micronuclei, CAs and SSBs were not significantly elevated in the workers. The activity of the u-isoenzyme of glutathione-S-transferase (GT) was measured as a possible genetic marker of susceptibility and a modulator of biomarker formation. However, possibly because of confounding by age, no significant relationships were found between GT and any of the exposure-related markers by ANOVA or among other independent variables by regression. This study demonstrates significant effects of low-level EtO exposure, independent of smoking history, near or below 1 ppm on multiple biomarkers and suggests that the current OSHA standard may not be adequately protective. Previously described effects of smoking on EtO-Hb adducts, SCEs and SCEHFC were also seen in this study.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell and microsome mediated binding of 7,12-dimethylbenz(a)anthracene to DNA studied by fluorescence spectroscopy.

Fluorescence spectra of DNA isolated from hamster embryo cells incubated with 7,12-dimethylbenz(a)anthracene, or DNA modified in a microsomal system by reaction with this carcinogen or its 7-hydroxymethyl derivative, were compared to various model compounds. The spectra indicate that the DMBA derivative bound to DNA, in all 3 cases, has a 9,10-dimethylanthracene-like chromophore. They also provide the first evidence of the similarity in structure of the DNA-bound products between 7,12-dimethylbenz(a)anthracene and its 7-hydroxymethyl derivative. Our results are consistent with an activation mechanism that involves saturation of the 1,2,3,4-ring positions.

DNA modification by chemical carcinogens

The chemistry and molecular biology of DNA adducts is only one part of the carcinogenic process. Many other factors will determine whether a particular chemical will exert a carcinogenic effect. For example, the size of particles upon which a carcinogenic may be adsorbed will influence whether or not, and if so where, deposition within the lung will occur. The simultaneous exposure to several different agents may enhance or inhibit the metabolism of a chemical to its ultimate carcinogenic form (Rice et al., 1984; Smolarek and Baird, 1984). The ultimate carcinogenic metabolites may be influenced in their ability to react with DNA by a number of factors such as internal levels of detoxifying enzymes, the presence of other metabolic intermediates such as glutathione with which they could react either enzymatically or non-enzymatically, and the state of DNA which is probably most heavily influenced by whether or not the cell is undergoing replication or particular sequences being expressed. Replicating forks have been shown to be more extensively modified than other areas of DNA. Another critical factor which can influence the final outcome of the DNA damage is whether or not the modifications can be repaired. If this occurs with high fidelity and the cell has not previously undergone replication then the effect of the damage by the carcinogen is likely to be minimal. The major area in which progress is needed is an understanding of what this damage really does to the cell such that after an additional period of time, which may be as long as twenty or more years, these prior events are expressed and cell proliferation occurs. Clearly additional stimulatory factors, for example tumor promoting agents such as the phorbol esters or phenobarbital, are often needed. After such prolonged periods it seems likely that the DNA adducts would no longer be present. However, the way in which their earlier presence is remembered is not clear. Simple mutations do not explain all the characteristics of tumor progression and, when it occurs, regression. Even if a specific site mutation does occur then its expression must be under other types of control. Any explanation of the action of DNA modification at the molecular level also requires that account be taken of the diverse nature of the DNA adducts from simple modifications such as methylation to bulkier adducts such as benzo[a]pyrene, aflatoxin or aromatic amines.(ABSTRACT TRUNCATED AT 400 WORDS)

Biologic Markers in Risk Assessment for Environmental Carcinogens

The potential of biologic markers to provide more timely and precise risk assessments for environmental carcinogens is viewed against the current state-of-the-art in biological monitoring/molecular epidemiology. Biologic markers such as carcinogen-DNA adducts and oncogene activation are currently considered valid qualitative indicators of potential risk, but for most chemical exposures research is needed to establish their validity as quantitative predictors of cancer risk. Biologic markers have, however, already provided valuable insights into the magnitude of interindividual variation in response to carcinogenic exposures, with major implications for risk assessment.

Ring test for low levels of N-(2-hydroxyethyl)valine in human hemoglobin

Hemoglobin adducts are useful for the identification and quantification of electrophilic agents in vivo. A modified Edman degradation method has been extensively used for monitoring exposure to ethylene oxide through gas chromatographic-mass spectrometric measurements of hydroxyethyl adducts to the N-terminal valines in hemoglobin. In a ring test, four laboratories using different versions of the method analyzed eight human globin samples with low adduct levels from ethylene oxide. Measurements of the same adduct by a radioimmunoassay were also included. Strong correlation between the measurements by the different laboratories shows that the method in principle works well. However, there were some systematic quantitative differences.

High-pressure liquid chromatography of carcinogen-nucleoside conjugates: separation of 7,12-dimethylbenzanthracene derivatives.

Abstract Conditions for obtaining rapid, high resolution separations of nucleic acid derivatives of 7,12-dimethylbenzanthracene using reverse phase high-pressure liquid chromatography are described. Direct reaction of dimethylbenzanthracene-5,6-oxide with poly(G) or poly(A) yields four and two major dimethylbenzanthracene-nucleoside conjugates, respectively. Incubation of [3H]dimethylbenzanthracene with poly(G) in a microsomal system leads to the formation of numerous guanosine derivatives, most of which differ from the 5,6-oxide products. The high sensitivity and resolution obtained in these studies demonstrate that high-pressure liquid chromatography will be a valuable technique for identification and characterization of other carcinogen-nucleic acid complexes.

Isotopic separations of tritium-substituted compounds from protium analogues by high-pressure liquid chromatography

Abstract The use of high-pressure liquid chromatography (HPLC) for the separation of a large variety of compounds is now common. Those compounds which contain hydrophobic moieties can often be well separated on reverse phase columns where the stationary hydrocarbon phase is bound to the support and the compounds are eluted with water, mixed with polar organic solvents such as methanol. One of the advantages of HPLC is the high column efficiency which can be obtained. Despite this, however, it may still not be possible to resolve certain closely related compounds, such as the 12 phenols of benzo[ a ]pyrene (BP) (1,2). Thus, when identity between a radioactive component and its unlabeled analogue is to be claimed, great care must be exercised. We were concerned, therefore, when, during an investigation (3) of products formed by the enzyme-catalysed binding of 7,12-[G- 3 H]dimethylbenz[ a ]anthracene (DMBA) to polyguanosine, some of the modified nucleoside adducts showed very slightly shorter retention times than the unlabelled compound to which it was thought they corresponded. the possibility that this was an isotopic separation (4) was investigated.

Photoaddition of chlorpromazine to DNA

Chlorpromazine, 2-chloro-N-(3-dimethylaminopropyl)phenothiazine (CPZ), is a frequently prescribed antipsychotic drug that causes cutaneous photosensitivity in man. CPZ is also phototoxic and photomutagenic in vitro. We have investigated the photoaddition of CPZ to DNA as a possible mechanism for these photobiologic effects. Prior to irradiation, CPZ binds non-covalently to double-stranded calf thymus DNA. At high nucleotide to CPZ ratios, the CPZ absorption maximum shifts from 305 nm to 340 nm with an isosbestic point at 323 nm and 90% of the CPZ fluorescence at 455 nm is quenched. The excitation and emission spectra for the unquenchable fluorescence are the same as those for unbound CPZ. The absorption and fluorescence spectra of unbound CPZ are restored at 0.1 mM magnesium acetate or 100 mM sodium acetate. Non-covalent binding of CPZ to heat-denatured DNA does not shift the CPZ absorption spectrum but quenches 65% of the CPZ fluorescence. Photolytic decomposition of CPZ was inhibited by binding to DNA. In the presence of high concentrations of double-stranded DNA or denatured DNA the photolysis rates were reduced by greater than 98% and 65%, respectively, compared to free CPZ. Formation of covalent photoadducts between CPZ and denatured DNA was 10-fold more efficient than photoadduct formation with double-stranded DNA. Approximately 10% of the CPZ which photodecomposed upon irradiation at 323 nm photoadded to denatured DNA. These results indicate that formation of a complex between CPZ and double-stranded DNA absorbing at 340 nm protects CPZ from photodecomposition and inhibits covalent photoadduct formation.

Mutagenicity of some methylated benzo[a]pyrene derivatives

The mutagenicity of benzo[a]pyrene (BP) and a number of methylated derivatives towards Salmonella typhimurium has been tested. The most mutagenic derivative tested was 6-methylbenzo[a]pyrene which produced about twice the number of revertants as did BP, 11-Methylbenzo[a]pyrene was slightly more mutagenic than BP. All the other compounds tested (7-, 8-, 9- and 10-methylbenzo[a]pyrene and 7,8- and 7,10-dimethylbenzo[a]pyrene) were significantly less active than benzo[a]pyrene. With the exception of 6-methylbenzo[a]pyrene, these results closely parallel the known carcinogenicity of the methylated benzo[a]pyrenes, and support the view that metabolic activation of BP may involve the 7-10 positions which are blocked in the methylated compounds.

Covalent binding of ethylated analogs of 7,12-dimethylbenz[a]anthracene to the DNA of mouse embryo fibroblast 10T12 cells

The carcinogenicity of a number of ethylated analogs of 7,12-dimethyl-benz[a]anthracene (DMBA) has been previously studied. The covalent binding of these compounds to mouse embryo fibroblast 10T1/2 cell DNA expressed as adducts/10(6) bases, paralleled the original carcinogenicity studies and in decreasing order of potency were DMBA, 7-ethyl-12-methylbenz[a]anthracene (7-Et,12-MeBA), 7-methyl-12-ethylbenz[a]anthracene (7-Me,12-Et), and 7,12-diethylbenz[a]anthracene (7,12-DEBA). The results demonstrate that a major determinant of the carcinogenicity of these compounds relates to their ability to bind to DNA.