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The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydrocarbons (PAH) were significantly higher in Athens subjects during both seasons. There was hardly any diagonal radioactive zone in the pattern of DNA adducts observed. Highest adduct levels were observed in a sub-group of subjects living in or near the Halkida Institute campus, which was located in rural surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution.

Groups of lambda lacZ transgenic mice were treated i.p. with N-nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in various tissues. Adduct induction in the liver exceeded by at least one order of magnitude than observed in the next nearest target tissue (lung), and was approximately linearly related to dose, except for O6-methylguanine after the first dose of 1 mg/kg which was lower than expected. Substantial induction of lambda lacZ mutagenesis was observed only in the liver, where the mutant frequency was already maximal within 7 days after 5 mg/kg NDMA and remained unchanged thereafter up to 49 days. Small but marginally significant increases in mutant frequency were consistently observed in the spleen after all three modes of treatment. A lack of proportionality between mutation induction and the administered dose or the corresponding adduct levels was observed, probably reflecting the importance of toxicity-related cell proliferation caused by NDMA at higher doses. Twenty eight days after a dose of 10 mg/kg (causing a 3.6-fold increase in mutant frequency), NDMA was found to increase the frequency of GC--AT mutations (with a concomitant shift of their preferential location from CpG sites to GpG sites), which made up approximately 60% of the induced mutations. Surprisingly, NDMA also caused a significant increase in deletions of a few (up to 11) base-pairs (22%).

The DNA damaging and mutagenic activities of procarbazine, a methylating drug employed in cancer chemotherapy and suspected of causing therapy-related leukaemia, were investigated in the liver and bone marrow of lambda lacZ transgenic mice (MutaMouse). The drug was administered using two different protocols, a 'high-dose' one involving 5 daily doses of 200 mg/kg, expected to cause depletion of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) and thus favour the selective accumulation of the premutagenic lesion O6-methylguanine (O6-meG) relative to other adducts, and a 'low-dose' one involving 10 daily doses of 20 mg/kg procarbazine. Substantial accumulation of O6-meG was observed in both tissues examined 6 h after the end of the 'high-dose' treatment, with the liver accumulating somewhat higher levels than the bone marrow (28.0 +/- 1.8 fmol/microg DNA and 18.5 +/- 1.1 fmol/microg DNA respectively). However, significant increases in mutant frequency 10 days after the end of treatment were observed only in the bone marrow, reaching a 16-fold increase over background following the 5 x 200 mg/kg treatment. Sequence analysis of the mutations induced after this treatment revealed a mixed spectrum, in which G:C-->A:T transitions (characteristic of O6-meG miscoding) were only a secondary feature: Among 20 mutants analysed, only six such mutations were found, including three at CpG sites, which might have arisen from deamination of 5-methylcytosine. The other mutations observed included 1 A:T-->G:C transition, five transversions (one G:C-->T:A, one double G:C-->C:G, two A:T-->T:A, one A:T-->C:G), five deletions and three insertions. The mechanistic and clinical significance of these findings is discussed.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of secondary biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involved in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose response and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity. Chemicals/CAS: Antineoplastic Agents, Alkylating; Blood Proteins; Carcinogens, Environmental; DNA Adducts; Epichlorohydrin, 106-89-8; Ethylene Oxide, 75-21-8; Methylene Chloride, 75-09-2; Mutagens; Nitrogen Oxides; Petroleum; Polycyclic Hydrocarbons, Aromatic; Styrene, 100-42-5; Styrenes

DNA repair is an important mechanism of cellular protection from the effects of genotoxic chemicals. Although extensive evidence from studies in experimental systems indicates that variation in DNA repair can significantly influence susceptibility to genotoxins, corresponding studies in human populations are so far limited, mainly because of methodological difficulties. One system, using observations of the accumulation and repair of DNA damage in cancer patients treated with alkylating cytostatic drugs, has provided useful information for assessing the effects of interindividual variation in DNA repair activity on the induction of genotoxic effects in humans. The most detailed studies of this kind have been carried out on patients with cancer (i.e., Hodgkin disease, malignant melanoma) treated with the methylating cytostatic drugs procarbazine or dacarbazine; these studies have provided detailed information on dose-response relationships. They have also demonstrated the protective role of the repair enzyme O6-alkylguanine-DNA alkyltransferase against the accumulation of the premutagenic methylated DNA lesion O6-methylguanine in patients' DNA. Given the strong evidence that exposure of the general population to environmental methylating agents may be extensive, as indicated by the frequent discovery of methylated DNA adducts in human DNA, data on DNA damage and repair in alkylating drug-treated patients and their modulation by host factors may prove useful in efforts to assess the possible carcinogenic risks posed by exposure to environmental methylating agents.

The kinetics of accumulation of the premutagenic DNA adduct O6-methylguanine (O6-meG) in the liver, blood leukocytes, lymph nodes and bone marrow of rats was examined and compared after single or multiple doses of procarbazine, a methylating cytostatic drug employed in the treatment of Hodgkin's lymphoma patients, and methylnitrosourea (MNU), an experimental methylating agent and carcinogen. Maximal O6-meG levels occurred 1-2 h after administration of single doses of procarbazine (10 mg/kg) or MNU (1 mg/kg), thereafter decreasing with half-lives of approximately 20-45 h, depending on the tissue. A relatively uniform tissue distribution was observed with both agents, with the liver generally showing highest adduct levels, followed by the lymph nodes, bone marrow and blood leukocytes which contained broadly similar amounts of O6-meG. During daily, oral administration to rats of procarbazine for 10 days at dose rates of 2.5, 5, 10 or 20 mg/kg/day (treatment analogous to that of the MOOP chemotherapy protocol for Hodgkin's lymphoma) followed by animal death on different days (in each case 24 h after the last treatment), a biphasic mode of O6-meG induction was observed: an initially steep build-up during the first 3-4 days was followed by a transient decline in the rate of accumulation, in turn followed by a second wave of accumulation and then a further slow-down. During the same treatment, liver O6-methylguanine-DNA alkyltransferase (AGT) declined in a dose-related manner. AGT recovery after the end of treatment was slow, taking nearly 20 days after the end of the high-dose treatment to return to control levels, despite the fact that all detectable adducts had been lost from DNA within 3 days after the end of treatment. A similar depletion and slow recovery of AGT in the liver, blood lymphocytes, bone marrow and lymph nodes was observed after treatment with a single dose of 100 mg/kg procarbazine. In contrast to these observations, O6-meG accumulated smoothly during a 10 day administration of MNU (1 or 10 mg/kg/day) to reach a steady-state within 5-6 days, while liver AGT was partially depleted after the high dose and recovered fully within 72 h of cessation of treatment. Similarly, a single dose of MNU (35 mg/kg) resulted in AGT depletion followed by rapid recovery in all four tissues examined. It is concluded that procarbazine (but not MNU) causes a decrease in cellular AGT concentrations by a mechanism additional to suicide repair of O6-meG.(ABSTRACT TRUNCATED AT 400 WORDS)

The accumulation of O6-methylguanine (O6-meG) in the DNA of blood leukocytes of 21 Hodgkin's lymphoma patients (followed for up to 12 cycles of treatment) treated in the context of MOPP combination chemotherapy with 150 mg procarbazine daily for 10 days was examined and compared to that observed in rats treated with different doses of procarbazine as a single agent once per day for 10 days. In humans, the adduct accumulated in a dose-related fashion and appeared to approach a steady-state after 7-8 days of treatment. Adduct levels on day 10 of the treatment cycle averaged 0.25 +/- 0.09 (mean +/- SD) mumol/molG and, for different individuals, covered a 3-fold range. Intra-individual variability between different treatment cycles was much more limited than inter-individual variability, the two parameters accounting for 8.9% and 84.5% respectively of adduct variance at a constant cumulative dose. Comparison of the dose-response relationships for humans and rats indicates that, under conditions of no depletion of O6-alkylguanine-DNA alkyltransferase (AGT), O6-meG accumulates in blood leukocyte DNA of humans at a rate which is only approximately 2-fold lower than in rats, implying that, to the extent to which O6-meG contributes to the genotoxic activity of procarbazine, human susceptibility to it is likely to be comparable to that of the rat. This is likely to be true also of the bone marrow (the tissue of interest as a target tissue for leukaemogenesis), since the tissue distribution of O6-meG induced by low doses of procarbazine in rats, mice and rabbits indicated that blood leukocyte levels of this adduct closely reflect those in the bone marrow. Based on these results, it is estimated that by the end of a MOPP chemotherapy cycle O6-meG reaches levels of the order of 0.2-0.3 fmol/microgram DNA (0.3-0.5 mumol/molG) in human bone marrow (the target tissue of leukaemogenesis observed after such treatment).

The first or/and the second guanines of the human Ha-ras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV40-based shuttle vector able to replicate in both simian cells and bacteria. After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E. coli DH5 alpha cells. The vast majority of the mutations induced by O6 meG were G----A transitions. The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% +/- 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% +/- 0.6). This difference was confirmed by the results obtained when two adjacent O6 meG residues were positioned within codon 12. The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity. These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.

Blood leukocyte DNA obtained from 11 Hodgkin's disease patients undergoing ABVD chemotherapy was analysed for the presence of the precarcinogenic adduct O6-methylguanine (O6-meG) at various times (1-2 h up to 49 h) after i.v. treatment with the methylating drug dacarbazine. Adduct formation was detected in all but one of the patients examined at levels ranging up to 0.45 fmol/micrograms DNA (7.2 x 10(-7) mol/mol guanine). The levels of the adduct decreased by approximately 30% over the 24 h following exposure and were usually not detectable 49 h after exposure. In five out of seven individuals examined after more than one treatment, consistent methylation responses were noted, while in the remaining two cases the responses were mixed. No correlation between the extent of adduct formation and lymphocyte levels of the repair enzyme O6-alkylguanine-DNA alkyltransferase was observed. The average extent of O6-meG formation 1 h after dacarbazine treatment was (4.3 +/- 3.1) x 10(-2) fmol/micrograms DNA per mg/kg dose [( 1.2 +/- 0.8) x 10(-3) fmol/micrograms DNA per mg/m2 dose)]. Following exposure of rats to similar doses of dacarbazine, the corresponding levels of adduct in blood leukocyte DNA were 1.1 x 10(-2) fmol/micrograms DNA per mg/kg dose (2.6 x 10(-3) fmol/micrograms DNA per mg/m2 dose).

The concentrations of nitrite, thermo- and acetic acid-labile TEA-responsive compounds (TACs) and N-nitroso compounds (NOCs) as a group were measured in human gastric juice collected just before and 1, 2 and 4 h after oral ingestion of 1 g ascorbic acid (AA) or 200 mg sodium nitrate, separately or in combination. Individual responses of gastric [nitrite] following ingestion of AA alone varied widely, with both decreases and increases being observed, and showed no correlation with gastric pH. While a mixed response was also noted for [NOC] and [TAC], substantial decreases were observed in 5/6 individuals with initial [NOC] greater than 0.2 microM and 3/3 individuals with initial [TAC] greater than 0.2 microM, implying that (i) AA effectively inhibited gastric nitrosation and (ii) a basal amount of NOCs and TACs was present in gastric juice which could not be lowered by AA ingestion. Statistical analysis indicated that global mean values of gastric [NOC] were significantly reduced (P less than 0.02) 1-4 h after ingestion of AA. Ingestion of 200 mg sodium nitrate alone resulted in increases in gastric [NOC], which in some cases were very substantial. While nitrosation appeared lower following ingestion of the same dose of nitrate in combination with 1 g AA, the difference from the effects of nitrate alone was not statistically significant. In aqueous buffer, pH 2.5, and in the presence of 1 mM AA, 50 microM nitrite was consumed with a t1/2 of 50 min only if molecular oxygen had first been removed from the system. In the presence of oxygen, no consumption of nitrite could be detected in 50 min, reflecting nitrite recycling (oxidation of nitric oxide to higher oxides of nitrogen and hydrolysis back to nitrite). It is likely that nitrite recycling occurring after collection of gastric juice accounted for the inconsistent responses of gastric nitrite following ingestion of AA. Incubation of human gastric juice, pH 2.5, in vitro in the presence of 50 microM sodium nitrite for 60 min resulted in an increase of [NOC] and [TAC] from 0.10 to 0.70 and 1.10 microM respectively. Nitrosation was efficiently inhibited by AA, 2.27 mM AA resulting in 87 and 100% inhibition respectively. Removal of oxygen from the reaction mixture did not have any significant effect on the extent of nitrosation in the presence or absence of AA.

DNA extracted from biopsies of normal or atrophic gastric mucosa obtained from 20 individuals was analysed for the presence of the precarcinogenic alkylation lesion O6-methylguanine by a recently developed, highly sensitive assay based on repair by the Escherichia coli O6-alkylguanine-DNA alkyltransferase (AGT) enzyme in competition with a radiolabelled oligonucleotide containing O6-methylguanine (O6-meG). With a limit of detection of 0.5 fmol O6-meG in 10 micrograms DNA, only one DNA sample (derived from a region of the stomach with advanced chronic atrophic gastritis) was found marginally positive, containing 0.52 fmol/10 micrograms DNA (8.3 X 10(-8) mol O6-meG/mol guanine). Measurements of AGT in 49 biopsies of normal, atrophic, hyperplastic or dysplastic mucosa obtained from the gastric antrum or corpus of 18 individuals did not reveal any significant effects of mucosal histology on AGT. The average AGT value found was 6.9 +/- 3.5 (SD) fmol/micrograms DNA, which is lower than the values reported for a number of other human tissues (liver, small intestine and lung). Measurement of AGT levels in gastric mucosa and circulating lymphocytes of the same individuals revealed a positive correlation (P less than 0.005), suggesting that lymphocytes may serve as a useful surrogate marker for AGT activity in gastric mucosa in studies of the epidemiology of this important repair enzyme.

The concentrations of nitrate, nitrite, N-nitroso compounds and bacteria were measured in 96 samples of fasting gastric juice, pH 0.90-8.50, obtained from 56 individuals just before or at various times (8 days - 1 year) after gastric operation. The mean pH of the post-operative samples [4.66 +/- 0.39 (SEM)] was significantly higher than that of the pre-operative ones [3.29 +/- 0.33 (SEM)]. A positive correlation with pH was observed for the concentrations of total and nitrate-reducing bacteria (median values 5.0 X 10(5) organisms/ml and 9.2 X 10(4) organisms/ml, respectively, for samples with pH greater than or equal to 1.2 X 10(3) organisms/ml and 0 organisms/ml, respectively, for samples with pH less than or equal to 2.5) and nitrite [mean values 22.5 +/- 3.1 (SEM) microM and 3.20 +/- 0.5 (SEM) microM for samples with pH greater than or equal to 6.5 and pH less than or equal to 2.5, respectively]. No correlation with pH was seen for the concentrations of nitrate [mean value 0.48 +/- 0.06 (SEM) mM] or N-nitroso compounds [mean value 0.30 +/- 0.06 (SEM) microM]. The concentrations of bacteria and nitrite, although increased in hypochlorhydric individuals, were lower than those reported for corresponding individuals in other, primarily British, studies. It is suggested that the relatively low concentrations of nitrite observed in our hypochlorhydric population may account for the absence of elevated concentrations of N-nitroso compounds and that the latter phenomenon may be related to the relatively low frequency of gastric cancer in Greece.

A simplified and highly sensitive assay for the determination of O6-alkylguanine-DNA-alkyltransferase has been developed and validated by the analysis of extracts of human urinary bladder mucosa. The new assay involves the use of a synthetic dodecanucleotide containing a single O6-methylguanine residue as substrate for the enzyme. This substrate is 5'-end-labelled with [35S]PO3 and separation of repaired and unrepaired oligonucleotide is achived by immuno-precipitation with polyclonal antibodies specific for O6-methyldeoxyguanosine. Kinetic analysis of the repair of the oligonucleotide by extracts of Escherichia coli and rat liver showed that the reaction is first-order in substrate and enzyme and gave the molecular rate constants 7.5 x 10(6) mol-1 1-1 sec-1 and 8.0 x 10(6) mol-1 1-1 sec-1, respectively. The rate constants for the repair of the corresponding O6-ethylguanine-containing oligonucleotide were 3.0 x 10(5) mol-1 l-1 sec-1 and 3.6 x 10(6) mol-1 l-1 sec-1, respectively. Analysis of extracts of 48 samples of normal or neoplastic human urinary bladder mucosa obtained by transurethral biopsy or at surgery, by the new method or by a method involving use of [3H]-methylated DNA as substrate and HPLC, indicated excellent agreement between the two methods. The mean AGT content of normal urinary bladder mucosa obtained from individuals without diagnosed bladder cancer was 0.181 +/- 0.081 (mean +/- SD) fmol/microgram protein, that of neoplastic samples 0.323 +/- 0.177 fmol/microgram protein and that of normal tissue obtained from patients with bladder cancer 0.183 +/- 0.068 fmol/microgram protein. The new method is convenient, rapid and extremely sensitive (it can readily measure femtomole quantities of enzyme) and should prove useful for studies of the biochemical epidemiology of DNA repair.

Fasting human gastric juice was treated in vitro, at pH 2-7 and 37 degrees C for 2 h, with 5-100 microM sodium nitrite. Under these conditions (which simulated those occurring in vivo in normal or hypochlorhydric individuals), the formation of total N-nitroso compounds had the following characteristics: (i) it increased greatly at pH less than 3; (ii) it showed first-order dependence on nitrite concentration; (iii) it was faster at pH 7 than at pH 5. These observations are compatible with the N-nitroso compounds formed by the interaction of nitrite with gastric juice being N-nitrosamides or related compounds. Furthermore, based on the results of this study, it is suggested that in order for hypochlorhydria to give rise to increased formation of N-nitroso compounds in the stomach, it would be necessary for it to be accompanied by a greater than 5- to 10-fold increase in gastric nitrite concentration relative to that found in the normal population, a condition which is not necessarily fulfilled in all hypochlorhydric individuals or populations. The implications of this conclusion for the assessment of the role on gastric N-nitroso compounds in the etiology of gastric cancer are discussed.