Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid.

It has been hypothesized that oxygen radicals generated by peroxidation of dietary linoleic acid may induce genetic damage and thereby increase cancer risk. We examined the effect of dietary supplementation with linoleic acid on the levels of oxidative DNA damage in peripheral lymphocytes and on the blood plasma antioxidant potential. Thirty volunteers received during 6 weeks either a high dose of linoleic acid (15 g/day), an intermediate dose of linoleic acid (7.5 g/day) or an isocaloric supplement without linoleic acid (15 g palmitic acid/day). After the intervention, no significant increase in oxidative DNA damage, measured as relative amounts of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in DNA from peripheral lymphocytes, was observed in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05). Also, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group receiving palmitic acid (23% decrease) were not significant. Furthermore, no statistically significant differences were found between the total antioxidant capacities of blood plasma from the different experimental groups. Plasma levels of malondialdehyde, an important end-product of lipid peroxidation, were not increased after supplementation, nor were effects found on the plasma concentrations of retinol, alpha-tocopherol and beta-carotene. Despite the experimental design that excludes several forms of bias introduced in studies based on modulation of dietary composition, our results provide no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid. Therefore, we see no scientific basis to reconsider the public health policy to stimulate the intake of polyunsaturated fatty acids aimed at the reduction of coronary heart diseases.

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms.

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.

Induction of DNA adducts by several polychlorinated biphenyls.

It is known that lower-chlorinated biphenyls are metabolically activated to electrophilic quinoid species capable of binding to DNA. Also, certain metabolites are capable of redox cycling, thereby increasing oxidative stress in biological systems. In the present study, we tested mono-, di-, tri-, tetra-, penta-, hexa-, and heptachlorinated biphenyls for their ability to bind with DNA and to induce oxidative DNA damage. We present additional evidence that several PCB congeners form DNA adducts after metabolic activation, which can be detected by the nuclease P1- or butanol-enrichment procedures of the (32)P-postlabeling technique. Butanol and nuclease P1 enrichments showed different adduct recoveries, depending on the level of chlorination of the biphenyls. Application of the nuclease P1 enrichment showed that the incubation of 2-chloro-; 3, 4-dichloro-; 2,4,4'-trichloro-; 3,4,5-trichloro-; and 2,2',5, 5'-tetrachlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital, followed by oxidation with a peroxidase, produced five to eight different DNA adducts. For these lower-chlorinated biphenyls, butanol enrichment generally showed a lower recovery. For some higher substituted congeners (3,3',4,4', 5-pentachloro-, 2,2',3,4,4',5'-hexachloro-, 2,2',4,4',5, 5'-hexachloro-, and 2,2',3,4,4',5,5'-heptachlorobiphenyl), after butanol enrichment a single dominant spot was observed, which was absent in the nuclease P1 procedure. After incubation of calf thymus DNA with either higher- or lower-chlorinated PCB congeners, we were not able to detect significantly increased levels of oxidative DNA damage above background levels, measured as 8-oxo-7, 8-dihydro-2'deoxyguanosine. In view of the carcinogenicity of PCB mixtures in animals and the ability of PCB metabolites to bind covalently to DNA, rats were orally treated with a mixture of PCBs (Aroclor 1242). PCB-DNA adduct levels were analyzed in PCB target organs: liver, thymus, glandular stomach, spleen, testes, seminal vesicles and prostate DNA. In vivo PCB-DNA adducts could not be detected by either the butanol- or by the NP1-enrichment procedure in rat target tissue DNA. Also, no differences in oxidative DNA damage could be observed between PCB-treated rats and controls. These results indicate a lack of DNA reactivity of PCB mixtures in vivo.

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons.

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.

Use of crayfish in biomonitoring studies of environmental pollution of the river Meuse.

The river Meuse, located in western Europe, is contaminated by different pollutants, of both organic and inorganic nature. The predominant sources of Meuse contamination in The Netherlands are agricultural activities and pollution derived from urban areas. Crayfish, water, and sediment samples were collected at four different locations of the river Meuse, in order to cover a large part of the catchment area of this river in The Netherlands. Crayfish may be very useful in biomonitoring studies, since they can integrate body load by pollutants over time in an area-bound manner. In these crayfish, levels of aromatic DNA adducts, heavy metal residues, polychlorinated biphenyls (PCBs), and organochlorine pesticides were determined in hepatopancreatic tissue. Also analyzed were water and sediment samples derived from the same locations, for polycyclic aromatic hydrocarbons (PAHs), heavy metals, and organochlorine compounds. In sediments from the four different sampling sites, no clear differences were observed in PCB levels. Organochlorine pesticide concentrations were highest at location A, the most upstream sampling site, whereas a general decrease was observed following the river Meuse downstream. A similar pattern was observed for the metal compounds. For PAH sediment levels no consistent tendency could be observed. Highest values were detected at site B, followed by, respectively, locations A, D, and C. In water samples, a different pattern was observed. The highest metal concentration was observed at location D, whereas the total organochlorine level was higher at sites B and D, compared to the two other sampling sites. Differences in pollution levels in crayfish between sampling sites were evident. Site D, the most downstream-situated site examined, appeared to be the most polluted site with respect to PCBs, DDT, DDE, and Cu in crayfish. Moreover, DNA adduct levels, which may serve as a dosimeter for the internal dose of aromatic compounds such as PAHs and PCBs, were also significantly higher in hepatopancreatic tissue of crayfish captured at site D, compared to the three other sampling sites. Moreover, significant correlations were observed between DNA adduct levels and the lower chlorinated PCB congeners (PCB 28-PCB 101). By correlating the different pollutants in water and/or sediment with xenobiotic levels in crayfish, no consistency could be observed, indicating that monitoring aquatic species may provide specific information on the presence of surface water pollutants. These results indicate that crayfish can be used as biological indicators of exposure to both organic and inorganic pollution in aquatic systems.

In vitro effects of coal fly ashes: hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells.

Oxygen radical generation due to surface radicals, inflammation, and iron release has been suggested as the mechanism of adverse effects of quartz, such as emphysema, fibrosis, and carcinogenic effects. Therefore, we measured iron release, acellular generation of hydroxyl radicals, and oxidative DNA damage and cytotoxicity in rat lung epithelial (RLE) cells by different coal fly ashes (CFA) that contain both quartz and iron. Seven samples of CFA with different particle size and quartz content (up to 14.1%) were tested along with silica (alpha-quartz), ground coal, and coal mine dust (respirable) as positive control particles, and fine TiO(2) (anatase) as a negative control. Five test samples were pulverized fuel ashes (PFA), two samples were coal gasification (SCG) ashes (quartz content <0.1%), and one sample was a ground coal. No marked differences between SCG and PFA fly ashes were observed, and toxicity did not correlate with physicochemical characteristics or effect parameters. Stable surface radicals were only detected in the reference particles silica and coal mine dust, but not in CFA. On the other hand, hydroxyl radical generation by all fly ashes was observed in the presence of hydrogen peroxide, which was positively correlated with iron mobilization and inhibited by deferoxamine, but not correlated with iron or quartz content. Also a relationship between acellular hydroxyl radical generation and oxidative DNA damage in RLE cells by CFA was observed. Differences in hydroxyl radical generation and oxidative damage by the CFA were not related to iron and quartz content, but the respirable ashes (MAT023, 38, and 41) showed a very extensive level of hydroxyl radical generation in comparison to nonrespirable fly ashes and respirable references. This radical generation was clearly related to the iron mobilization from these particles. In conclusion, the mechanisms by which CFA and the positive references (silica, coal mine dust) affect rat lung epithelial cells seem to be different, and the data suggest that quartz in CFA does not act the same as quartz in silica or coal mine dust. On the other hand, the results indicate an important role for size and iron release in generation and subsequent effects of reactive oxygen species caused by CFA.

Neutrophils cause oxidative DNA damage in alveolar epithelial cells.

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H2O2, cause oxidative DNA damage in epithelial cells.

Analysis of oxidative DNA damage and HPRT mutant frequencies in cancer patients before and after radiotherapy.

Various markers of radiation-induced DNA damage including DNA oxidation were investigated in peripheral lymphocytes of 23 cancer patients prior to and one week after receiving radiotherapy with a cumulative dose of 54-70 Gy. Exposure to ionizing radiation nonsignificantly increased the ratio 2'deoxy-7-dihydro-8-oxoguanosine/2'deoxyguanosine (8-oxodG/dG) from 1.73 x 10(-5) to 3.33 x 10(-5). Frequencies of micronuclei significantly (p = 0.0003) increased from 6.4 to 38.9 per 1000 cells. The frequency of hypoxanthine-guanine-phosphoribosyltransferase (HPRT) mutant lymphocytes measured as 6-thioguanine resistant variant cells by 5-bromodeoxyuridine labeling, was elevated eight-fold, from 4.7 x 10(-6) to 36.2 x 10(-6) (p = 0.008) after termination of the radiotherapy, thus showing a clear response to the radiation treatment. No correlation between levels of oxidative DNA damage and frequencies of HPRT mutant lymphocytes or micronuclei could be established.

Oxidative DNA damage and cytogenetic effects in flight engineers exposed to cosmic radiation.

This study set out to analyze biomarkers for genotoxic events, e.g., oxidative DNA damage, chromosomal damage and hprt mutations, among flight personnel, who are known to be occupationally exposed to ionizing radiation of cosmic origin. Twenty-three flight engineers were recruited while ground personnel served as a matched control group. Cumulative radiation doses during flight were calculated on the basis of subjects' flight records assuming an exposure rate of 6 microSv per hour of flight. Oxidative DNA damage in peripheral lymphocytes from flight engineers appeared significantly increased in comparison with controls and was associated with cumulative exposure to cosmic radiation. Frequencies of peripheral lymphocyte chromosome aberrations, micronuclei and hprt mutations appeared also to be increased in flight engineers, but not significantly. It was also observed that DNA damage was higher in flight engineers with a relatively shorter flight history in comparison with flight engineers with higher cumulative exposures to radiation, suggesting adaptation to DNA damage caused by ionizing radiation. DNA repair activities measured as unscheduled DNA synthesis were clearly increased in the higher-exposed subgroup of flight engineers, and appeared significantly correlated with cumulative radiation dose, as well as inversely with oxidative DNA damage. The implications for cancer risk assessment in relation to exposure to cosmic radiation are discussed.

Comparison of 32P-postlabeling and HPLC-FD analysis of DNA adducts in rats acutely exposed to benzo(a)pyrene.

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Possible relevance of pigeons as an indicator species for monitoring air pollution.

Wild city pigeons were caught at four different locations in the Netherlands to represent areas of high (Amsterdam-high), moderate (Amsterdam-medium), and low (Maastricht and Assen) traffic density. It is assumed that local ambient air pollution decreases as a function of traffic density. In these pigeons levels of polycyclic aromatic hydrocarbon (PAH)-DNA adducts, oxidative DNA damage, and heavy metal residues were determined in kidney, lung, liver, and blood (no adduct analysis in blood). The contribution of leaded gasoline to total body lead content was estimated by measuring concentrations of Pb and its isotopes in blood. We also analyzed samples of ambient air particulate matter for PAH and heavy metal concentrations at the four different locations. Interregional differences in heavy metals in ambient air particulate matter were reflected relatively well by pigeon body loads. The higher lead and cadmium concentrations in blood, kidney, liver, and lung were found in the Amsterdam high traffic density area, followed by Amsterdam medium, Assen, and Maastricht. A high Pb concentration in blood coincided with relatively low 206Pb/207Pb values, indicating a high contribution of leaded gasoline to total blood Pb concentrations in pigeons from the Amsterdam high traffic density area. Significantly enhanced blood zinc values were found in pigeons from both locations in Amsterdam compared to pigeons from the other two areas. However, no differences in Zn tissue levels between the four different groups were found. Oxidative DNA damage, determined as the ratio of 7-Hydro-8-oxo-2'-deoxyguanosine/ deoxyguanosine, in pigeon liver was highest in Amsterdam-high, followed by Assen (low traffic density). Pb content, but not the Cd content, was positively associated with oxidative DNA damage in liver tissue. In lung tissue, a negative correlation was found between oxidative DNA damage and Zn content. These results indicate that the carcinogenic potential of Pb might be ascribed to oxygen radical formation, whereas Zn plays a protective role against oxidative DNA damage. Places with high and medium traffic density could be clearly discriminated on the basis of PAH levels in the ambient air. The PAH content in particulate air samples was not, however, reflected in total PAH-related DNA adduct levels because no differences could be observed in tissue adduct levels in pigeons from the four different locations. Our results indicate that wild city pigeons can be used as biological indicators of exposure to heavy metal pollution in outdoor air.

Bioavailability of soil-adsorbed cadmium in orally exposed male rats.

During the last few decades, the industrial production and use of Cd resulted in the release of significant quantities of Cd into the environment. Concern about health risks of human exposure to this toxic metal, which may be contained in soil and other environmental compartments, has increased significantly in recent years. Soil ingestion is a potentially important pathway of exposure to soil-absorbed environmental contaminants, especially for young children exhibiting hand-to-mouth behavior. Health risk assessments are usually based on unchanged bioavailability of soil-absorbed pollutants, e.g., heavy metals, neglecting interactions of metals with the soil matrix, which may lead to relatively lower bioavailability. This study was conducted to determine the bioavailability of Cd absorbed to soil in rats. Eight-week-old male Lewis rats were given either a soil polluted with CdCl2 (150 micrograms Cd/rat) dissolved in 5% gun acacia or an equal amount of Cd as CdCl2 dissolved in saline. Control rats were gavaged with isotonic saline. Cd concentrations in liver, kidney, brain, heart, and blood, as well as Cd content of urine and feces were analyzed using graphite furnace atomic absorption spectrometry. Tissue Cd concentrations in soil-treated animals were significantly lower than the tissue concentrations in the Cd-saline group; in the liver and kidneys of the Cd-saline and Cd-soil groups, 4 and 2.7% respectively, of the original doses were recovered. Relative bioavailability, calculated on the basis of blood Cd levels for the Cd-soil group as compared to the Cd-saline group, appeared to be 43%. No differences in the excretion pattern of Cd into feces were observed between the Cd-saline and Cd-soil groups. After 6 days, over 91% of the original dose was recovered in the feces of both Cd-treated groups. Cd excretion via urine was very low, but in the Cd-soil group a significant increase in urinary Cd was observed as compared to the control group. However, the amount of Cd excreted into urine of the Cd-soil group during the experimental period corresponded to only 0.01% of the original dose. In the Cd-saline group, no additional Cd was excreted into urine as compared to the control group. These results indicate that the soil matrix significantly reduced the absorption of Cd in the gastrointestinal tract. Consequently, exposure assessment models, assuming an unaffected bioavailability of soil-absorbed Cd, overestimate the internal dose and thereby overestimate health risks associated with direct ingestion of soil particles.

Reducing effects of garlic constituents on DNA adduct formation in human lymphocytes in vitro.

A water extract of raw garlic (RGE) and two organosulfur compounds, diallyl sulfide and S-allylcysteine (SAC), were evaluated for their relative effectiveness in reducing benzo[a]pyrene (BaP)-DNA adduct formation in stimulated human peripheral blood lymphocytes in vitro. In replicate experiments, RGE significantly inhibited BaP-DNA adduct formation at concentrations of 0.001, 0.01, and 0.1 mg/ml. SAC also significantly decreased BaP-DNA adduct formation at concentrations of 0.01 and 0.1 mg/ml. For diallyl sulfide, no significant reduction in BaP-DNA adduct formation was found. BaP-DNA adduct formation was not associated with cell viability or proliferation of peripheral blood lymphocytes after the various treatments. No clear scavenging activity was detected for the garlic constituents. Aryl hydrocarbon hydroxylase activity was not decreased, nor was formation of sulfate and glucuronide conjugates of 3-hydroxy-BaP increased in the presence of RGE and SAC, indicating that increased glutathione S-transferase activity or a more efficient repair of BaP-DNA adducts may explain the observed effects. In addition, reactive oxygen species-induced 8-oxodeoxyguanosine in DNA was reduced in the presence of SAC. It is concluded that raw garlic and SAC may be useful in the prevention of BaP-associated tumorigenesis and that further evaluation of their preventive potential in humans at risk appears feasible.

Induction of oxidative DNA damages and enhancement of cell proliferation in human lymphocytes in vitro by butylated hydroxyanisole.

The food additive butylated hydroxyanisole (BHA) has been shown to induce gastrointestinal hyperplasia in rodents by an unknown mechanism. The relevance of this observation for human risk assessment is not clear. We therefore analysed the effect of BHA and its primary metabolites tert-butylhydroquinone (TBHQ) and tert-butylquinone (TBQ) on 8-oxo-deoxyguanosine formation and labelling induces in human lymphocytes in vitro. Analysis of culture medium and cell lysate fractions after administration of BHA or metabolites of BHA revealed that BHA and TBHQ undergo biotransformation in whole blood cultures. Moreover, TBQ can be reduced to TBHQ. While in cultures treated with BHA 50-60% of the dose administered was recovered, a much lower dose recovery was found in cultures treated with either TBHQ or TBQ. This indicates a considerable binding of these compounds to macromolecules. BHA and TBHQ, as well as TBQ, induced a dose-dependent increase in cell proliferation of phytohaemagglutinin-stimulated lymphocytes, 50 microM being the optimal dose. Since BHA is metabolized to TBHQ, it is not clear which compound is responsible for the proliferation enhancing effects observed in culture. Inhibition of IBHQ metabolism to its semiquinone radical by acetylsalicylic acid (ASA) reduced the increase in labelling indices induced by TBHQ. This indicates that this metabolic pathway is involved in the enhancement of cell proliferation induced by the hydroquinone. HPLC-ECD analysis of oxidative DNA damage in lymphocytes exposed to 10, 50 and 100 microM BHA, TBHQ or TBQ respectively showed that BHA was not capable of inducing oxidative DNA damage to a significant degree. TBQ and, in particular, TBHQ at a dose of 50 microM (the optimal dose for induction of cell proliferation), however, increased lymphocyte 7-hydroxy-8-oxo-2'-deoxyguanosine formation by 320 and 680% respectively. Inhibition of prostaglandin H synthase by ASA in cultures treated with TBHQ decreased the oxidation ratio significantly, confirming the significance of this enzyme system in the mechanism of toxicity of BHA.

Induction of oxidative DNA damage and early lesions in rat gastro-intestinal epithelium in relation to prostaglandin H synthase-mediated metabolism of butylated hydroxyanisole.

The effect of metabolic activation of the food additive 3-tert-butyl-4-hydroxyanisole (BHA) by prostaglandin H synthase on the gastro-intestinal cell proliferation was determined by studies of the nature and the time dependency of early lesions in the forestomach, glandular stomach and colon/rectum of rats given BHA with and without co-administration of acetylsalicyclic acid (ASA: an inhibitor of prostaglandin H synthase), in combination with the formation of oxidative DNA damage in the epithelial cells of glandular stomach and colon/rectum as well as in the liver. BHA appeared to be a strong inducer of oxidative DNA damage in the epithelial cells of the glandular stomach, increasing the level of 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) with increasing duration of BHA administration. Similar observations were made in colorectal DNA although levels of oxidative DNA damage tend to be smaller. In liver DNA, BHA appeared to be capable of increasing background 8-oxodG levels only after 14 days of treatment. This relatively slow response may be related to very low prostaglandin H synthase activity of liver cells. The severity of hyperplasia and inflammation in both forestomach and glandular stomach appeared to increase gradually with continued BHA administration. The hyperplasia induced by BHA was paralleled by inflammatory changes. In colorectal tissue, however, no tissue abnormalities were observed. This indicates that oxidative DNA damage induced by BHA is not a consequence of early lesions in gastro-intestinal epithelium, but might be the initial step in the stimulation of gastro-intestinal cell proliferation which, as shown previously, also occurs in colon epithelium. Co-administration of the prostaglandin H synthase inhibitor ASA resulted in a significant decrease of both epithelial oxidative DNA damage and the incidence of lesions, which indicates that this enzyme system is involved in the enhancement of cellular proliferation induced by BHA. Co-oxidation by prostaglandin H synthase of the BHA-metabolite tert-butylhydroquinone into tert-butylquinone, yielding active oxygen species, might therefore be responsible for the carcinogenic effects of this food antioxidant.

Oxidative DNA damage in peripheral blood lymphocytes of coal workers.

Reactive oxygen species are important mediators of both mineral dust-induced (malignant) lung disease and in vitro DNA damage. Therefore, we studied in vivo oxidative DNA damage in coal workers who had been chronically exposed to silica-containing dust. In peripheral blood lymphocytes of 38 retired coal miners (eight with coal workers pneumoconiosis, 30 references) and 24 age-matched, non-dust-exposed controls 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was determined by reversed phase high-performance liquid chromatography with electrochemical detection. The ratio of 8-oxodG residues to deoxyguanosine (dG) was related to individual cumulative dust exposure estimates and pneumoconiotic stage as established by chest radiography. The ratio of 8-oxodG to dG(x 10(-5)) in lymphocytes did not differ between miners with coal workers' pneumoconiosis (2.61 +/- 0.44) and miners without coal workers' pneumoconiosis (2.96 +/- 1.86). However, oxidative DNA damage in all miners was higher than in the non-dust-exposed controls (1.67 +/- 1.31). 8-oxodG/dG ratio was not related to individual cumulative coal dust exposure, age or smoking (pack years) when evaluated by multiple linear regression. We suggest that oxidative damage to the DNA of peripheral blood lymphocytes may be introduced by increased oxidative stress responses in subjects chronically exposed to mineral dusts. Whether this is an important pathway in the suggested carcinogenicity of silica is still an open question.

The role of prostaglandin H synthase-mediated metabolism in the induction of oxidative DNA damage by BHA metabolites.

The carcinogenicity of the phenolic food antioxidant butylated hydroxyanisole may be related to its oxidative biotransformation in vivo. In order to determine the ability of BHA, 2-tert-butyl(1,4)hydroquinone (TBHQ) and 2-tert-butyl(1,4)paraquinone (TBQ) to induce oxidative DNA damage, biological inactivation of single-stranded bacteriophage phi X-174 DNA, as well as induction of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in dG by these compounds was studied in vitro, in the presence and absence of peroxidases. Both test systems showed that BHA and TBQ (probably due to lack of reductase activity in vitro) were not capable of inducting oxidative DNA damage. TBHQ, however, appeared to be a strong inactivator of phage DNA as well as a potent inducer of 8-oxodG formation. Addition of radical scavengers showed that this damage was due to formation of superoxide anion, hydrogen peroxide and hydroxyl radicals. Addition of iron chelators and metal ions showed that the one-electron oxidations of TBHQ via the semiquinone radical into TBQ are toxic via the formation of oxygen radicals and are not directly due to the hydroquinone itself or the formation of semiquinone radicals. Although peroxidation of TBHQ by prostaglandin H synthase (PHS) is indicated to result in a superoxide anion burst, this is not accompanied by an increase in oxidative DNA damage in vitro. This might be due to the use of hydrogen peroxide as a substrate by PHS itself, consequently resulting in less formation of hydroxyl radicals. Oxidation of TBHQ by lipoxygenases showed that no semiquinone radicals or oxygen radicals were formed, probably due to a two-electron oxidation of TBHQ directly into TBQ. The present results indicate that metabolic activation of BHA yielding reactive oxygen species may induce a carcinogenic potential, since the BHA metabolite TBHQ, appeared to be a strong inducer of oxidative DNA damage.

Oxygen radical formation during prostaglandin H synthase-mediated biotransformation of butylated hydroxyanisole.

The dominant metabolic pathway of the presumably carcinogenic food antioxidant 2(3)-tert-butyl-4-hydroxyanisole (BHA) includes O-demethylation to 2-tert-butyl(1,4)hydroquinone (TBHQ) and subsequent peroxidation to 2-tert-butyl(1,4)paraquinone (TBQ). In order to determine the ability of TBHQ to induce the formation of oxygen radicals, electron spin resonance measurements were performed in presence and absence of peroxidases. ESR analyses showed that prostaglandin H synthase resulted in a substantially accelerated metabolism of TBHQ into TBQ, which is accompanied by formation of superoxide anion, hydroxyl radical and hydrogen peroxide. Spectrophotometric measurements revealed that prostaglandin H synthase and lipoxygenase are both capable of converting TBHQ into TBQ. In order to determine the effect of prostaglandin H synthase on BHA (dose-level: 1.5% BHA of the diet) metabolism in vivo, we coadministered two inhibitors of prostaglandin H synthase acetylsalicylic acid and indomethacin, with BHA to rats. Coadministration of acetylsalicylic acid (0.2%) in the drinking water resulted in a significant increase of urinary TBHQ excretion. Both acetylsalicylic acid and indomethacin (dose-level: 0.002% in the drinking water) induced a significant decrease in TBQ excretion into urine. Co-oxidation by prostaglandin H synthase of the BHA-metabolite TBHQ into TBQ, yielding reactive oxygen species might therefore be responsible for the carcinogenic and toxic responses elicited by this antioxidant.

Effects of butylated hydroxyanisole on arachidonic acid and linoleic acid metabolism in relation to gastrointestinal cell proliferation in the rat.

In order to determine the effect of oral administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA; dose-level: 1.5% BHA of the diet) on arachidonic acid (AA) and linoleic acid (LA) metabolism in correlation with changes in gastrointestinal cell kinetics, we coadministered two inhibitors of prostaglandin H synthase, acetylsalicylic acid (ASA) and indomethacin (IM), to rats. Coadministration of ASA (0.2%) and IM (0.002%) in the drinking water, resulted in a significant reduction of the BHA-induced enhancement of cell proliferation in forestomach and glandular stomach. ASA completely counteracted the effect of BHA on labeling indices in colon/rectum whereas IM exhibited no effect in this organ. Both inhibitors had no direct effect on cell kinetics in the control groups. ASA, and to a lesser degree IM, inhibited prostaglandin E2 release in all tissues examined. Whereas ASA did inhibit lipoxygenase-mediated metabolism of AA in forestomach tissue, ASA did not affect the release of AA- and LA-derived hydroxy fatty acids in glandular stomach and colon/rectum. IM did not affect lipoxygenase production. BHA, however, appeared to be a strong inhibitor of both routes of AA metabolism. While ASA nor IM affected LA metabolism, BHA inhibited both prostaglandin H synthase-mediated and lipoxygenase-mediated metabolism of AA and LA. A causal role of AA or LA metabolites in the process of cell proliferation enhancement induced by BHA, can therefore be excluded. Prostaglandin H synthase may, however, be involved in BHA activation by converting the hydroquinone metabolite of BHA to the corresponding quinone by redox cycling, which is probably accompanied by reactive intermediate production.

Modulation by dietary factors of BHA-induced alterations in cell kinetics of gastro-intestinal tract tissues in rats.

To determine the effects of dietary ethanol or fibre on 2(3)-tert-butyl-4-hydroxyanisole (BHA)-induced alterations in cell kinetics in gastro-intestinal tract tissues, groups of six male Wistar rats were fed diets containing 0% (control) or 1.5% BHA for 2 wk. One group fed 1.5% BHA and one pair-fed control group received 10% ethanol in the drinking-water; two similarly fed groups received drinking-water only. Another group fed 1.5% BHA and a pair-fed control group received a diet supplemented with 20% cellulose; two similar groups received no fibre supplementation. Cell kinetics in the forestomach, glandular stomach and oesophageal tissue were determined, after 14 days, by bivariate 5-bromo-deoxyuridine/DNA analysis using immunocytochemistry and flow cytometry. In the fibre experiment, colorectal tissue was also examined. In both experiments the labelling indices in all the gastro-intestinal tract tissues were significantly altered in the BHA-fed groups compared with the corresponding control groups. In the ethanol experiment no statistically significant difference in the labelling indices was observed in the forestomach or glandular stomach between the two control groups or between the two BHA-fed groups. However, intake of ethanol-supplemented drinking-water induced increases in oesophageal labelling indices in rats fed a BHA-free diet. Thus 14 days of simultaneous ethanol administration has no effect on BHA-induced alterations in cell kinetics in the oesophagus, glandular stomach or forestomach of rats. In the forestomach and colorectal tissue, a high-cellulose diet resulted in a significant decrease in the BHA-induced elevation of labelling indices. Thus dietary cellulose provides a partial protection against the proliferation-enhancing effects of BHA in the rat gastro-intestinal tract.