Genetic polymorphisms and micronucleus formation: a review of the literature.

The formation of micronuclei (MN) is extensively used in molecular epidemiology as a biomarker of chromosomal damage, genome instability, and eventually of cancer risk. The occurrence of MN represents an integrated response to chromosome-instability phenotypes and altered cellular viabilities caused by genetic defects and/or exogenous exposures to genotoxic agents. The present article reviews human population studies addressing the relationship between genetic polymorphisms and MN formation, and provides insight into how genetic variants could modulate the effect of environmental exposures to genotoxic agents, host factors (gender, age), lifestyle characteristics (smoking, alcohol, folate), and diseases (coronary artery disease, cancer). Seventy-two studies measuring MN frequency either in peripheral blood lymphocytes or exfoliated cells were retrieved after an extensive search of the MedLine/PubMed database. The effect of genetic polymorphisms on MN formation is complex, influenced to a different extent by several polymorphisms of proteins or enzymes involved in xenobiotic metabolism, DNA repair proteins, and folate-metabolism enzymes. This heterogeneity reflects the presence of multiple external and internal exposures, and the large number of chromosomal alterations eventually resulting in MN formation. Polymorphisms of EPHX, GSTT1, and GSTM1 are of special importance in modulating the frequency of chromosomal damage in individuals exposed to genotoxic agents and in unexposed populations. Variants of ALDH2 genes are consistently associated with MN formation induced by alcohol drinking. Carriers of BRCA1 and BRCA2 mutations (with or without breast cancer) show enhanced sensitivity to clastogens. Some evidence further suggests that DNA repair (XRCC1 and XRCC3) and folate-metabolism genes (MTHFR) also influence MN formation. As some of the findings are based on relatively small numbers of subjects, larger scale studies are required that include scoring of additional endpoints (e.g., MN in combination with fluorescent in situ hybridization, analysis of nucleoplasmic bridges and nuclear buds), and address gene-gene interactions.

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

Kinetics of the formation and removal of cisplatin-DNA adducts in blood cells and tumor tissue of cancer patients receiving chemotherapy: comparison with in vitro adduct formation.

During chemotherapy with a cisplatin-containing combination of drugs, 217 blood samples from 30 cancer patients were analyzed for the presence of the main cisplatin-DNA adduct cis-Pt(NH3)2d(pGpG) (Pt-GG). Cisplatin was administered during 3-h infusions on each of 5 consecutive days, resulting in increasing adduct levels which, on the average, were about twice as high after the fifth as after the first infusion. Higher levels were found in blood samples of patients who received the same total amount of cisplatin in one single 3-h infusion. No significant differences in adduct levels were found during first and repeated courses. The nonlinear dependence of adduct levels on total dose can be attributed to removal of adducts. At 21 h after a very first cisplatin infusion 76% of the adducts were removed. Lower percentages of removal were observed over the 21-h periods following the fourth and fifth infusions of 5-day courses (49 and 53%, respectively). After the initial 21 h the removal of adducts continued, albeit at a slower rate. Substantial interindividual variation was found in the adduct levels, which did correlate with the levels obtained after in vitro cisplatin treatment of blood samples from the same patients but not with their age or gender. Testicular cancer patients with complete tumor response showed higher adduct levels in their blood than those with partial response or progressive disease. When blood samples from 8 healthy volunteers were treated with cisplatin in vitro, the person-to-person variation in adduct levels and the intraindividual variation observed over a 2-year period were found to be in the same range, which was narrower than that observed with samples from treated patients. In vitro studies with human blood showed that the formation of the Pt-GG adduct is proportional to cisplatin concentration and complete after about 1 hour. In some of the in vivo and in vitro cisplatin-treated blood samples, all 4 known platinum-DNA adducts were determined. In all cases Pt-GG was by far the major adduct, and no significant differences were observed with respect to the relative amounts of the 4 adducts. Similar adduct ratios were found in DNA from a testicular tumor obtained from a patient who underwent orchidectomy; the Pt-GG adduct level was about 10-fold higher than that in his blood cells.

Detection of photorepair of UV-induced thymine dimers in human epidermis by immunofluorescence microscopy.

To investigate the effect of visible light on the level of UV-induced thymine dimers in human epidermal cells in vivo, we exposed volunteers to UV-B alone, or to a serial combination of UV-B and visible light. Dimers were assayed in skin sections by immunofluorescence microscopy with a monoclonal antibody against the cyclobutyl thymine dimer. The dimer-specific fluorescence from epidermal cell nuclei, identified by counterstaining with propidium iodide, was quantified through computer-mediated image processing and analysis. After a single UV exposure (2-3 MED), significant dimer-specific fluorescence was measured, but no difference could be detected between skin kept in the dark after UV-irradiation and that exposed to visible light. In three other experiments, the UV dose was split into 3 parts (1 MED each), given at 2.5-h intervals. Half of the skin area was exposed to visible light following each dose fraction. After the second and third dose fractions, skin areas treated with visible light clearly showed lower levels of dimers (i.e., about 40% reduced) than skin kept in the dark. The results provide evidence that photorepair of dimers does occur in human skin, but not immediately after a first UV exposure of naive skin.

Detection of thymine dimers in suprabasal and basal cells of chronically UV-B exposed hairless mice.

An immunocytochemical method was developed to study induction and removal of DNA damage in specific cell populations in the epidermis of hairless mice during chronic ultraviolet (UV) exposure. Identification of mouse suprabasal cells was performed with an immunoperoxidase stain. This stain was shown not to affect the fluorescent nuclear stains, used to reveal DNA and DNA damage. In skin cells from hairless mice irradiated daily with 1500 J/m2 UV-B for 11 consecutive days, cyclobutane thymine dimers accumulated in epidermal cells and reached a maximum level after 3 d. Thereafter dimer levels dropped to a lower, more constant level. So epidermal cells in vivo, both suprabasal and basal cells, remove dimers effectively, in contrast to cultured rodent cells, which display hardly any repair in genomic DNA. Dimer content in suprabasal cells was higher than that in basal cells, but initially the patterns of induction and removal of dimers in both cell types were rather similar. At days 4-11, however, after the drop in dimer content, the amount of dimers in basal cells prior to UV exposure was almost as low as that in non-exposed cells. The results presented here suggest important roles for both UV-induced DNA repair and cell proliferation in protecting epidermal cells against the mutagenic and carcinogenic effects of UV.

Evaluation of the carcinogenic risks to humans associated with surgical implants and other foreign bodies - a report of an IARC Monographs Programme Meeting. International Agency for Research on Cancer.

A meeting was held within the International Agency for Research on Cancer (IARC) Programme on the Evaluation of Carcinogenic Risks to Humans of surgical implants and other foreign bodies. This meeting report summarises the types of materials considered, their wear and degradation, their cancer epidemiology in both humans and other animals, the published experimental carcinogenicity data and selected data on their toxic, including genotoxic, effects. Evaluations resulting in a classification of Group 2B (possibly carcinogenic to humans) were reached for: (1) polymeric implants prepared as thin smooth films [with the exception of poly(glycolic acid)]; (2) metallic implants prepared as thin smooth films; and (3) implanted foreign bodies consisting of metallic cobalt, metallic nickel and a particular alloy powder consisting of 66-67% nickel, 13-16% chromium and 7% iron. Group 3 classifications (not classifiable as to their carcinogenicity to humans) were made for: (1) organic polymeric materials as a group; (2) orthopaedic implants of complex composition and cardiac pacemakers; (3) silicone breast implants; (4) dental materials; and (5) ceramic implants.

Immunochemical detection of DNA in alkaline sucrose gradient fractions.

A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.

Detection of DNA adducts in basal and non-basal cells of the hamster trachea exposed to benzo(a)pyrene in organ culture.

DNA adducts were quantified in hamster tracheas exposed to benzo(a)pyrene (BP) in organ culture, in basal as well as in non-basal cells, by in situ detection with an adduct-specific rabbit antiserum (W2/01) and with a mouse monoclonal antibody against human cytokeratins 5 and 8 (RCK102) to identify hamster trachea basal cells. Recognition by W2/01 of the adduct of (+)-anti-7,8-dihydroxy-9,10-epoxide of BP (BP-diolepoxide; BPDE) to deoxyguanosine (dG) was checked on human white blood cells (WBCs) exposed to BP together with 3-methylcholanthrene (3MC)-induced rat-liver microsomes. By comparison with the adduct levels determined by 32P post-labeling, a lower detection limit of about 1 adduct per 10(6) nucleotides could be deduced. Next, tracheal rings were exposed to BP (40 microM) in organ culture for 2 days, then washed and cultured without BP for another 3 days. At different time points epithelial cells were isolated and cytospin preparations made. Staining of BP DNA adducts combined with that of cytokeratin (both visualized with fluorescence) allowed detection of adducts in both basal and non-basal cells in the same preparation. BP DNA adduct formation in basal and non-basal cells after 2 days of exposure to BP was not different. However, on removal of BP the adducts disappeared significantly faster from basal cells than from non-basal cells. The combination of the two antibodies mentioned above thus allows selective determination of BP DNA adduct levels in different cell types. This could be of importance with regard to the involvement of specific cell types in the process of tumor initiation.

Methyl bromide causes DNA methylation in rats and mice but fails to induce somatic mutations in lambda lacZ transgenic mice.

Following single or multiple oral treatments of rats or lambda lacZ transgenic mice with methyl bromide, methylated DNA adducts (N7- and/or O6-methylguanine) were found at comparable levels in various tissues, including among others the glandular stomach, the forestomach and the liver. Multiple rat treatment resulted in substantial decreases in the repair enzyme O6-alkylguanine-DNA alkyltransferase which were probably due in part to direct interaction of the enzyme with methyl bromide. However, no induction of mutagenesis in the lacZ transgene could be detected in any tissue 14 days after single treatments of up to 50 mg/kg or after multiple treatments of as many as 10 daily treatments of 25 mg/kg MeBr.

Induction of somatic mutations but not methylated DNA adducts in lambdalacZ transgenic mice by dichlorvos.

In order to examine the in vivo genotoxic activity of dichlorvos, lambdalacZ transgenic mice (Muta Mouse) were treated i.p. with single (4.4 or 11 mg/kg) or multiple (5 x 11 mg/kg) doses of this agent and sacrificed 4 h or 14 days post-treatment for DNA adduct measurement or mutant frequency analysis, respectively. Neither methylated DNA adducts nor an increase in mutant frequency were detected in the bone marrow, white blood cells, liver, spleen, lung, brain and sperm cells after the single doses. However, following multiple dosing a statistically significant 3-fold increase in mutant frequency was observed in the liver, while a non-statistically significant increase was observed in the bone marrow. In contrast, dimethylsulphate, a model methylating agent, gave rise to detectable DNA adducts but no increase in mutant frequency following i.p. administration of single (30 mg/kg) or multiple (10 x 6 mg/kg) doses.

Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development.

Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. The formation of spermatozoa cisplatin-DNA adducts showed dose and time-dependent increases both in vitro, and in vivo up to 168 h (7 days) after dosing. Treatment of rats with 10 mg cisplatin/kg resulted in spermatozoa Pt-GG adduct levels of approximately 1.0 fmol/microg DNA. When cisplatin-treated male rats were bred to untreated females 6-24 h after cisplatin administration, no adverse developmental effects or decreases in body weight were seen in the offspring although there was a trend towards increased early embryo mortality.

Determining N7-alkylguanine adducts by immunochemical methods and HPLC with electrochemical detection: applications in animal studies and in monitoring human exposure to alkylating agents.

Many xenobiotics exert their toxic effects through interaction with DNA in the cells of the exposed organism. This interaction may lead to the formation DNA adducts. Some of these may give rise to mutations that initiate cell transformation and, ultimately, the formation of tumors. Sensitive methods for determining DNA adducts are indispensable for the study of chemical mutagenesis and carcinogenesis and for biomonitoring human exposure to genotoxic agents. Alkylating agents form an important class of genotoxic compounds. They react preferentially at the N7-position of guanine. Under neutral or acidic conditions, the adducts can be readily released from the DNA backbone as the free base N7-alkylguanine (N7-AlkGua). The imidazole ring of N7-alkyldeoxyguanosine (N7-AlkdGuo) can be opened under alkaline conditions, which results in formation of a more stable adduct in DNA. To develop immunochemical methods for the detection of N7-alkylations, we immunized mice with various alkylguanosines in the ring-opened form (RON7-AlkdGuo). Antibodies were selected to detect adducts in isolated DNA by competitive ELISA and in single cells by immunofluorescence microscopy (IFM). Various monoclonal antibodies were characterized in detail with respect to specificity and sensitivity toward methylated, ethylated, and hydroxyethylated DNAs. The antibodies showed extensive cross-reactivity toward N7-(m)ethyl- and N7-(2-hydroxyethyl)guanine modifications in the ring-opened form. The limits of detection in the direct and competitive ELISA were 5-10 and 1-2 adducts per 10(6) nucleotides, respectively. The detection limit of the IFM method was about 20 adducts per 10(6) nucleotides(ABSTRACT TRUNCATED AT 250 WORDS)

Use of monoclonal and polyclonal antibodies against DNA adducts for the detection of DNA lesions in isolated DNA and in single cells.

Interaction of genotoxic chemicals with their intracellular target, i.e., DNA, may result in the formation of covalent adducts. Various methods have been developed to estimate exposure to genotoxic chemicals by means of molecular dosimetry of DNA adducts. Such experiments have generally been carried out with radiolabeled genotoxicants administered in vitro to cultured cells or in vivo to laboratory animals. Biomonitoring of human exposure to genotoxic chemicals requires methods to detect very small quantities of nonradioactive DNA adducts in limited amounts of sample. Attention has been devoted to the development of immunochemical techniques in which specific DNA adducts can be detected with antibodies. The level of sensitivity achieved in these experiments renders these methods applicable for human biomonitoring. When suitable antibodies are available, the immunochemical approach enables one to analyze various types of adducts separately, and to discriminate between irrelevant (e.g., quickly repairable) and relevant lesions (key lesions) with respect to biological end points such as mutation induction and cancer. Polyclonal and monoclonal antibodies were used for the detection of DNA adducts in animal and human tissue. Adducts were measured in DNA from various organs of rats treated with the liver carcinogen 2-AAF. Human exposure to genotoxic agents was studied by the measurement of DNA adducts in blood cells from patients treated with the genotoxic cytostatic cisplatin. Also, the development is described of a system to detect and quantitate DNA adducts at the single-cell level by means of immunofluorescence microscopy, which allows the analysis of small samples of human tissue with preservation of cell morphology.

Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms.

Disease development in flounder (Platichthys flesus) was studied over a period of 3 years in three large mesocosms (40 m x 40 m x 3 m). Two of the mesocosms contained clean sand and the third, sharing a common water circulation with one of the clean-sand mesocosms, was stocked with contaminated dredged spoil. In this way, one of the clean-sand mesocosms was indirectly polluted via the water phase, and analysis of contaminant concentrations in sediments and flounder tissues showed that it had a status intermediate between the other two. Random samples of the flounder populations from the indirectly polluted and reference mesocosms were examined every 2 months for epidermal diseases (lymphocystis, skin ulcers, fin rot) and then released. In addition, every 6 months, random samples of fish from all three mesocosms were sacrificed for histological and chemical investigation. With regard to the development of epidermal disease, the results showed little difference between the reference mesocosm and the indirectly polluted mesocosm, with the exception that lymphocystis was significantly elevated in the indirectly polluted mesocosm. Although pollution may be a risk factor in the etiology of this disease, such a relationship would probably be obscured under field conditions due to variation arising from other factors. Histopathological analysis of the livers revealed in total four cases of hepatocellular adenoma (1.5% of sampled population) in fish from the polluted mesocosms, the first occurring after 2.5 years of exposure in fish from the indirectly polluted mesocosm. Furthermore, several other liver lesions, including foci of cellular alteration and hydropic vacuolated lesions, developed during the course of the experiment before tumor formation was apparent. Prevalences of these conditions were very much lower in the reference mesocosm than in the two polluted mesocosms. Densities of melanomacrophage centers in the liver showed a similar trend. The findings clearly indicate that long-term exposure to chemically contaminated dredged spoil can induce liver neoplasia and other liver lesions in flounder at contaminant levels comparable to those found in the natural environment.

32P-postlabelling analysis of DNA adducts in white blood cells of humans exposed to residential wood combustion particulate matter.

Residential wood combustion (RWC) in open fireplaces poses a possible health risk because of the emission into the indoor air of mutagenic and carcinogenic compounds. In the present report it was investigated whether this emission leads to enhanced levels of DNA adducts in white blood cells (WBC) of exposed subjects. Under conditions that most likely reflect the Dutch pattern of use of open fireplaces, RWC increased both indoor air mutagenicity and levels of benzo(a)pyrene (B(a)P) and pyrene. The indirect mutagenicity showed a stronger increase than the direct mutagenicity. The increase in indirect mutagenicity was not directly correlated with the increase in the levels of B(a)P and pyrene. 32P-postlabelling analysis of DNA adducts following nuclease P1 enrichment or butanol extraction revealed low adduct levels. No combustion-related increase in the amount of adducts was observed. Possible explanations for the lack of correlation between air monitoring data and WBC DNA adduct levels are discussed.

Influence of nucleotide excision repair on N-hydroxy-2-acetylaminofluorene-induced mutagenesis studied in lambda lacZ-transgenic mice.

To study the influence of nucleotide excision repair (NER) on mutagenesis in vivo, ERCC1 +/-, XPA-/-, and wild-type (ERCC1+/+ and XPA+/+, respectively) lambda lacZ-transgenic mice were treated i.p. with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and lacZ mutant frequencies were determined in liver. No significant effect of the treatment on the mutant frequency in wild-type or ERCC1-heterozygous mice was observed. The liver mutant frequency appeared to be significantly increased in treated XPA-/- mice only. To distinguish N-OH-AAF-induced from spontaneous mutations, lacZ mutants derived from treated XPA-/- mice were subjected to DNA-sequence analysis and the spectrum obtained was compared to that established for lacZ mutants in liver of PBS-treated lambda lacZ-transgenic mice of the parent strain 40.6. The N-OH-AAF-induced mutation spectrum appeared to be significantly different from the spontaneous mutation spectrum: the former consisted of mainly (19/22) single bp substitutions targeted at G, of which the majority (12/19) were G:C-->T:A transversions, suggesting that N-(deoxyguanosin-8-yl)-2-aminofluorene [dG-C8-AF], the major DNA adduct in N-OH-AAF-treated mice, is the premutagenic lesion. After analysis of 21 spontaneous mutants, only ten single bp substitutions targeted at G were found, of which five were G:C-->T:A transversions. This study with XPA-/- lambda lacZ-transgenic mice shows that one of the components of NER, that is, the XPA protein, suppresses mutagenesis in vivo.

UVB-induced mutagenesis in hairless lambda lacZ-transgenic mice.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (MutaMouse), which contain the lambda gt10lacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m2 UVB or to two successive irradiations of either 200 and 800 J/m2 UVB, with intervals of 1, 3, or 5 days, or to 800 and 200 J/m2 UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. The mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C --> A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C --> A:T transitions at CpG sites. The results indicate that the hairless lambda lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations.

DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating agents.

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Monitoring for DNA damage of humans occupationally exposed to methyl bromide.

BACKGROUND: Methyl bromide (MeBr) is a methylating agent, weak mutagen and possible animal carcinogen. A molecular epidemiological study to examine human exposure to, and consequent DNA damage by MeBr was conducted in an area where this agent is used extensively for soil sterilisation in greenhouses. MATERIALS AND METHODS: During the first part of the study, blood samples were collected from 21 persons within 24 hours after use of MeBr for greenhouse sterilisation, as well as from 19 non-exposed subjects. Personal air sampling was also carried out, indicating mean air concentrations for different subjects in the range 11-78 mg/m3. In the second part of the study, an attempt was made to examine professional applicators of MeBr who suffered particularly high exposures (mean exposures, based on personal monitoring 23-165 mg/m3). The levels of N7-methylguanine and O6-methylguanine, two DNA adducts known to be induced by MeBr, were assessed in blood leukocyte DNA. RESULTS: Concerning the first part, two subjects (one exposed and one control) were found to be positive for N7-methylguanine, while none of the blood samples analysed had detectable levels of O6-methylguanine. Among 6 such persons examined during the second part, 2 were found positive for N7-methylguanine while none was positive for O6-methylguanine. CONCLUSION: Within the detection power of this limited study, no significant evidence of induction of DNA damage in blood leukocyte DNA by MeBr was found.