DNA solution(R) in cigarette filters reduces polycyclic aromatic hydrocarbon (PAH) levels in mainstream tobacco smoke.

Tobacco consumption represents a major health hazard to humans and, despite anti-smoking campaigns, the number of smokers remains high; thus the reduction of toxic compounds from tobacco smoke may reduce the health hazards of smoking. In the last 25 years cigarette manufacturers have introduced a variety of filter designs to reduce toxic and carcinogenic substances in tobacco smoke (normal filters, NF). However, large quantities of harmful constituents are inefficiently retained by commonly used cigarette filters. Following a patented method we modified commercial cigarette filters (modified filter, MF) by injecting a DNA solution into the filter tips; we then evaluated the reduced polycyclic aromatic hydrocarbon (PAH) levels in mainstream tobacco smoke of MF relative to NF. The PAH measured were: fluoranthene (FLUO), pyrene (PY), benzo(a)anthracene (B(a)A), chrysene (CRY), benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene (B(k)F), benzo(g,h,i)perylene (BGP), dibenzo(a,h)anthracene (DBA). The levels of PAH in cigarette smoke after MF were significantly reduced (P<0.001) compared to NF, using a variety of cigarette brands in a smoking machine (44.5%+/-8.4 % and 41.8%+/-5% for total and carcinogenic PAH, respectively, means+/-SE). Using B(a)P(TEF) values the reduction in PAH concentrations were similar for all cigarette brands with the exception of Camel, where the reduction was lower considering B(a)P(TEF) values. Amongst carcinogenic PAH, B(a)A, B(b)F and B(k)F) were reduced by 50-58%, CRY, B(a)P and DBA by about 40%. In conclusion MF filters treated with DNA have the potential of decreasing the exposure to PAH in cigarette smoke. Since, unlike some previously proposed biological filters MF do not retain additional nicotine, the main addictive compound of tobacco smoke, these filters may not induce increased smoking to compensate for the reduction in the nicotine delivery to smokers.

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes.

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.

Urinary excretion of sucrose and fructose as a predictor of sucrose intake in dietary intervention studies.

The urinary excretion of sucrose, glucose, and fructose was measured in 9 healthy subjects consuming a common Italian diet and after 3 days of a low sucrose diet, in which the intake of sucrose was restricted but the other main nutrients were unmodified. After the low sucrose diet, we observed a significant drop in the average urinary excretion of sucrose, glucose, and fructose determined at four different times (8:00 and 10:00 a.m.; 3:00 and 10:00 p.m.). The average urinary excretion of fructose in the four urine samples was significantly correlated with dietary sucrose intake. We also found a significant correlation between the average urinary excretion of sucrose and dietary sucrose intake. Urinary fructose can be used as a marker of sucrose intake in dietary intervention studies aimed at studying the effect of variation of carbohydrate intake on specific cancers.

Urinary mutagens in humans after fried pork and bacon meals.

Urinary mutagenic activity on Salmonella typhimurium strain TA1538 with S9 was determined after morning meals of fried pork and bacon. Both in fasting and non-fasting subjects a very marginal elevation of urinary mutagenic activity was observed, accounting for a small fraction only of the total amount of mutagens ingested.

Dietary sucrose and starch affect dysplastic characteristics in carcinogen-induced aberrant crypt foci in rat colon.

To study whether dietary carbohydrates affect dysplasia in aberrant crypt foci (ACF), rats treated with 1,2-dimethilhydrazine (DMH) were fed for three months with diets containing 46% sucrose or corn starch. The number of ACF/colon in the two dietary groups was similar (P = 0.58), but ACF were smaller in the starch than in sucrose group (P < 0.05). ACF in the starch group also showed less severe goblet cell dysplasia, more sulphomucins and less sialomucins than in the sucrose group (P < 0.05), indicating that corn starch protects against colon carcinogenesis while sucrose in the diet is detrimental, promoting the dysplasia of preneoplastic lesions like ACF.

Release of free, redox-active iron in the liver and DNA oxidative damage following phenylhydrazine intoxication.

Following the subchronic intoxication of rats with phenylhydrazine, resulting in marked anemia, reticulocytosis, methemoglobinemia and increased hemocatheresis, the hepatic content of total iron was increased, as was hepatic ferritin and its saturation by iron. A striking increase (approximately 7-fold) was also observed in free iron which appeared to be redox-active. The increase in liver free iron involved the hepatocellular component of the liver. Since DNA is one of the cellular targets of redox active iron, liver DNA from phenylhydrazine-treated rats was analyzed by electrophoresis and found to be markedly fragmented. Experiments with isolated hepatocytes in culture or in suspension challenged with phenylhydrazine or Fe-nitrilotriacetate strongly suggested that the DNA damage was due to reactive iron rather than to the hepatic metabolism of phenylhydrazine. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a specific marker of oxidative DNA damage, were significantly higher in phenylhydrazine-treated rats as compared to untreated controls. The prolongation of phenylhydrazine treatment over a period of 6 weeks resulted in a persistent damage to DNA and in phenotypic changes such as an increase in hepatocyte gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2) activity. Possible relationships between iron overload, iron release, DNA damage and tumor initiation are discussed.

Protective effect of gangliosides on myocardial hypoxic damage in the rat.

The size of the infarct produced by ligation of the left coronary artery in the rat was decreased significantly in animals treated i.p. with 40 mg/kg per day of a ganglioside mixture (GMIX) for 7 days after surgery. Rats treated with GMIX had lower ventricular myeloperoxidase activity, indicating a lower leukocyte infiltration after infarction. The underperfused zone was also smaller in animals treated daily with GMIX 30 days after surgery. Control hearts, but not hearts obtained from animals pretreated for 15 days with 40 mg/kg per day of GMIX, released lactate dehydrogenase (LDH) during perfusion in a Langerdorff apparatus after ligation and reperfusion of the left coronary artery in vitro. Hearts made hypoxic in vitro by changing the perfusion gas to nitrogen for 20 min and later reoxygenating with 95% O2 -5% CO2 released LDH in the perfusate, but did not do so in the presence of 10 microM monosialotetraesosylganglioside. Gangliosides, therefore, seem to protect the rat heart against hypoxic damage.

Effects of GM1 ganglioside on cardiac function following experimental hypoxia-reoxygenation.

Rat hearts made hypoxic for 20 min by perfusion with 95% N2/5% CO2 and reoxygenated for 20 min in a Langerdorff apparatus showed a dose-dependent reduction of lactate dehydrogenase release when incubated with ganglioside GM1 (0.1-10 microM). The decline of contractile force during hypoxia was also reduced dose dependently in the presence of GM1. Similar effects were observed in hearts obtained from animals treated i.p. with 40 mg/kg GM1 for 14 days. The levels of Na+,K(+)-ATPase in ventricular tissue were also reduced after hypoxia-reoxygenation and the reduction was prevented in hearts from GM1-treated animals. GM1 (1-30 microM) reduced the functional response to field stimulation of adrenergic nerve terminals in isolated atria. Rat atria made hypoxic in glucose-free media maintained normal stores of tissue noradrenaline in the presence of 1 microM GM1. In the rabbit, GM1 (40 mg/kg i.p. for 4 days) reduced the alterations of the ST segment of the ECG during acute occlusion of the left descending and circumflex coronaries artery. In conclusion, ganglioside GM1 reduces some effects of hypoxia-reoxygenation in the heart, through still unknown mechanisms.

Oxidative liver DNA damage in rats treated with pesticide mixtures.

Oxidative damage was quantified in the liver of rats by measuring the levels of 8-OH-2-deoxyguanosine (8-OH-2DG) relative to 2-deoxyguanosine in DNA after treating rats for 10 days at a total dose of 1 mg/kg/day with a mixture of the 15 pesticides most commonly found in Italian foods (comprised of dithiocarbamate, benomyl, procymidone, methidathion, chlorpyrifos-ethyl, parathion-methyl, chlorpropham, parathion, vinclozolin, chlorfenvinphos, pirimiphos ethyl, thiabendazole, fenarimol, diphenylamine and chlorothalonil). We fractionated this pesticide mixture into subgroups in order to determine which molecules, if any, induced DNA oxidative damage. The administration of diphenylamine (0.09-1.4 mg/kg/day) and chlorothalonil (0.13-1 mg/kg/day) induced a dose-dependent increase in 8-OH-2DG levels in liver DNA. The other 13 pesticides of the mixture on the contrary, did not produce oxidative liver DNA damage. These results indicate that the toxicity of low doses of pesticide mixtures present in food might be further reduced by eliminating diphenylamine and chlorothalonil.

Benzo[a]pyrene diol-epoxide DNA adducts and levels of polycyclic aromatic hydrocarbons in autoptic samples from human lungs.

Benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) which are present in cigarette smoke, are common air and food genotoxic contaminants and possible human carcinogens. We measured the following PAH levels: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene, benzo[g,h,i]perylene as well as (+/-) syn and anti BaP diol-epoxide (BPDE) DNA adducts in autopsy samples from the lungs of non-smokers, ex-smokers and smokers who had lived in Florence, Italy. PAH levels in lung tissue were similar in all groups, with the exception of dibenzo[a,h]anthracene (DBA), which was higher in lung samples from smokers (n = 10, 0.18+/-0.17 ng/g d.w, mean +/- S.D.) compared to non-smokers (n = 15, 0.046+/-0.025 ng/g d.w) (P < 0.05), whereas ex-smokers (n = 5), had intermediate levels (0.07+/-0.03 ng/g d.w). The average level of total BPDE-DNA adducts was 4.46+/-5.76 per 10(8) bases in smokers, 4.04+/-2.37 per 10(8) in ex-smokers and 1.76+/-1.69 per 10(8) in non-smokers. The levels of non-smokers were significantly different (P < 0.05) from the levels of the smokers and ex-smokers combined. Total BPDE-DNA adducts were correlated with BaP levels in the lung samples in which both determinations were obtained (r = 0.63). Our results demonstrate that the biological load of PAHs due to environmental pollution is similar in individuals who smoke and those who do not, but BPDE-DNA adducts are higher in smokers and ex-smokers compared to non-smokers. This study further confirms the usefulness of BPDE-DNA adduct levels determination in the lungs from autopsy samples for monitoring long-term human exposure to BaP, a representative PAH.

In vitro DNA binding of 2-amino-3-methylimidazo(4,5-f)[5-3H]quinoline (IQ) is modulated by sulfotransferase and sulfate levels.

DNA binding in vitro was measured with 2-amino-3- methylimidazo(4,5-f)[5-3H]quinoline (3H-IQ) in the presence of S9 and microsomes from Wistar rat liver. DNA binding of 3H-IQ was catalyzed by both microsomes and S9 and was increased 5-fold in Aroclor (PCB)-pretreated animals. DNA binding was reduced by the specific inhibitor of sulfotransferase 2,6-dichloro-4-nitrophenol and slightly increased by 3'-phosphoadenosine 5'-phosphosulfate. The incubation of IQ in media with increasing concentrations of sulfate produced a modest dose-dependent increase in DNA binding. Liver S9 obtained from rats which had been administered 1% sulfite in drinking water catalyzed a higher binding of IQ to DNA, and this effect was inhibited by pentachlorophenol. The data demonstrate that sulfotransferase has a role in the activation of IQ and that variations in the activity of this enzyme induced by dietary changes may vary the genotoxic activity of IQ.

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations.

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.

Effect of a mixture of 15 commonly used pesticides on DNA levels of 8-hydroxy-2-deoxyguanosine and xenobiotic metabolizing enzymes in rat liver.

The level of 8-OH-2-deoxyguanosine in rat liver DNA was measured as an index of oxidative damage after treating rats for 10 days at a dose ranging from 0.75 to 10 mg/kg with a mixture of 15 pesticides (dithiocarbamate, benomyl, thiabendazole, diphenylamine, chlorthalonil, procimidone, methidathion, chlorpyrifos-ethyl, fenarimol, parathion-methyl, chlorpropham, parathion, vinclozolin, chlorfenvinphos, pirimiphos-ethyl) commonly found in foods of central Italy. At the doses of 0.75 and 1 mg/kg DNA levels of 8-OH-2-deoxyguanosine were significantly increased relative to controls, whereas at higher doses (2.5, 5, 10 mg/kg) the levels returned to control values. The administration of the pesticide mixture dose dependently reduced benzo(a)pyrene hydroxylase, N-demethylase activities, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and thiol transferase activities in the liver. The results show that the pesticide mixture induced free radical DNA damage at low doses. However, at higher doses it produced a depression of cellular metabolism, inhibiting a further expression of oxidative damage.

Effect of natural phenolic acids on DNA oxidation in vitro.

We examined the antioxidant activity of the following natural phenolic compounds present in food: 3-OH-benzoic acid (3-OH-BA); 4-OH-benzoic acid (4-OH-BA); 2,3-dihydroxybenzoic acid (2,3-diOH-BA); 3,4-dihydroxybenzoic acid (3,4-diOH-BA or protocatechuic acid); ferulic acid; caffeic acid; and 2-coumaric, 3-coumaric and 4-coumaric acids. We measured the inhibitory effect of these compounds on iron-dependent oxidative DNA damage in vitro [incubating herring sperm DNA with Fe(III)/GSH] or using cumene hydroperoxide (CumOOH) as a free-radical generating system; we also studied the interaction of these phenols with Fe(II) or Fe(III) spectrophotometrically. Among the tested compounds, 2,3-diOH-BA, 3,4-diOH-BA and caffeic acid interacted with Fe(II) and showed a potent inhibitory effect on iron-induced oxidative DNA damage. CumOOH-induced DNA oxidation was not modified by these compounds. On the contrary, 2-coumaric, 3-coumaric and 4-coumaric acids did not interact with iron but protected against oxidative DNA damage induced by Fe(III)/GSH and by CumOOH, indicating a direct free-radical scavenging activity of these compounds in both systems. The IC(50)+/-S.E.M. of the three coumaric acids against CumOOH-induced DNA oxidation was 44.2+/-2.0, 54.7+/-2.0 and 33.1+/-1.0 microM, respectively. On the contrary, 3-OH-BA and 4-OH-BA did not have scavenging activity and 3-OH-BA actually enhanced oxidative DNA damage. In conclusion, some natural phenolic acids, commonly present in food, have interesting protective activity against DNA oxidation in vitro and deserve further consideration as effective antioxidants in vivo.

Inhibition of 1,2-dimethylhydrazine-induced oxidative DNA damage in rat colon mucosa by black tea complex polyphenols.

The effect of black tea polyphenols on 1,2-dimethylhydrazine (DMH)-induced oxidative DNA damage in rat colon mucosa has been investigated. Fischer 344 rats were treated orally with thearubigin (TR) or theafulvin (TFu) for 10 days (40 mg/kg), injected ip with DMH (20 mg/kg) or saline and sacrificed 24 hr after DMH administration. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in colonic mucosa DNA and expressed as a ratio relative to 2'-deoxyguanosine (2dG). Control rat mucosa had 8-OHdG values of 1.12 +/- 0.14/10(5) dG (mean +/- SEM, n=11), whereas DMH-treated rats significantly higher values (1.52 +/- 0.14/10(5) dG, n=26, P<0.05). Pretreatment of rats with TR had significantly inhibited DMH-induced oxidative DNA damage 0.99 +/- 0.09/10(5) dG, n=10, P<0.05) and a similar, although less marked, effect was observed with TFu (1.15 +/- 0.19/10(5), n=9, P=0.06). These findings confirm that DMH causes oxidative DNA damage in the colon mucosa of rats and demonstrate that this effect is prevented by the consumption of complex polyphenols from black tea.

The polysaccharide from Tamarindus indica (TS-polysaccharide) protects cultured corneal-derived cells (SIRC cells) from ultraviolet rays.

The aim of this work was to investigate the possible protective effect of a new viscosising agent, TS-polysaccharide, on corneal-derived cells (SIRC) exposed to ultraviolet-B rays. To verify this, SIRC cells were first exposed, in the absence or in the presence of TS-polysaccharide (1% w/v), for 9 s at the UV-B source and then post-incubated for 45 min at 37 degrees C. After this period the hydrogen peroxide (H(2)O(2)) accumulated in the medium and the concentration of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) in cell DNA was measured. In addition, the amount of (3)H-methyl-thymidine incorporated in cellular DNA was evaluated after 18 h from irradiation. Our results show that cells exposed to UV-B rays accumulate H(2)O(2), and have higher levels of 8OHdG and a lower amount of (3)H-methyl-thymidine incorporated in DNA than control cells. In the presence of TS-polysaccharide, the H(2)O(2) and 8-OHdG accumulation, and the (3)H-methyl-thymidine incorporation were significantly reduced with respect to the values measured in cells exposed in the absence of the polysaccharide. We propose a protective role of the polysaccharide in reducing UV-B derived DNA damage to eye cells. This finding could be of some clinical importance when the polysaccharide is used as a delivery system for ophthalmic preparations.

Polycyclic aromatic hydrocarbons air levels in Florence, Italy, and their correlation with other air pollutants.

Benzo(a)pyrene [B(a)P] air levels were measured in Florence (Italy) in the period 1992-2001. For the period 1999-2000 seven polycyclic aromatic hydrocarbons (PAH) (benzo(a)anthracene, crysene, benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene, dibenzo(a,h)anthracene (DBA) and benzo(g,h,i)perylene (BGP)), were measured in the air in four different sites (one with heavy traffic (A), one in a park (B), one in a residential area (C) and one in a hill area (D)). B(a)P levels were elevated in 1992-1998 (maximum average value of winter months: 5.8 ng/ m3) but a decreasing trend was observed in the following years, probably due to improvement in vehicle emissions. The sum of PAH in the air in the period 1999-2000 was about one order of magnitude lower in the hill site (D) relative to the urban sites, and residential areas (B and C) had values 2.5-3 times lower compared to site A with a heavy traffic. PAH concentrations decreased in the warmer seasons of 2000 in all sites. A negative correlation was found between PAH levels and ozone. A positive correlation with carbon monoxide (CO) (r = 0.862, P < 0.001) and low B(a)P/BGP ratios, ranging from 0.44 to 0.51, indicated that vehicular traffic was the major PAH source in all monitored sites. Using B(a)P(TEF) values (toxic equivalency factors) for evaluating the biological activity of PAH, we found that the highest PAH contributors in terms of potential air carcinogenic activity were B(a)P and DBA. Therefore, in addition to B(a)P, DBA concentration should be considered in the evaluation of air quality in terms of PAH contamination.

Polycyclic aromatic hydrocarbons in Laurus nobilis leaves as a measure of air pollution in urban and rural sites of Tuscany.

The levels of 9 polycyclic aromatic hydrocarbons (PAHs) 6 of which carcinogenic were measured in the leaves of evergreen tree (Laurus nobilis) sampled in 13 locations in summer and winter in Tuscany, Italy. The carcinogenic PAH levels were correlated with the PAH air levels sampled at the same site. Samples from larger towns had higher PAH levels than those from medium and small towns. Leaves collected in the center of larger cities had higher carcinogenic PAH levels than samples from residential areas indicating that vehicular traffic is the main PAH source. Carcinogenic PAH levels in leaves collected in the winter in medium towns were considerably higher than expected, probably due to domestic heating. These findings demonstrate that air quality in terms of PAH contamination is not markedly different in towns of different size in Tuscany.

Lysyloxidase and benzylamine oxidase in pig aorta.

Three enzymes were isolated from pig aorta: a benzylamine oxidase differing from the plasma type, and two different types of lysyloxidase. Some properties of the two types of lysyloxidase are described. The three enzymes were inhibited by cupric copper chelating agents and by carbonyl reagents. They did not cross-react with the antibodies to pure pig plasma benzylamine oxidase.

[Levels of polycyclic aromatic hydrocarbons (PAHs) in air samples in the city of Arezzo (1997-1998)]. / Livelli aerei di idrocarburi policiclici aromatici (IPA) nella città di Arezzo (1997-1998).

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed environment pollutants of air, water and soil. Since many PAHs are potent mutagens and/or carcinogens the occurrence of these compounds in the lower atmosphere is an important element of environmental pollution. We measured PAH levels in airborne particles collected in the town of Arezzo, (Tuscany, Italy), during the period April 1997-February 1998. Air monitoring for 24 h was repeated for 7 days, during two weeks, in each season; a total of 84 air samples were obtained sampling two urban sites where the traffic is the main source of pollution. One site is a residential area. The data of this study indicate a pronounced seasonal variation in PAH levels and show that in cold spells other sources of contamination besides vehicular traffic are important.