Enzymatic biomarker measurement and study of DNA adduct formation in benzo[a]pyrene-contaminated mussels, Mytilus galloprovincialis

DNA adducts, benzo( a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

DNA adducts as markers of exposure to carcinogens and risk of cancer

Acute effects of benzo[a]pyrene on digestive gland enzymatic biomarkers and DNA damage on mussel Mytilus galloprovincialis

Previous studies by us and others have shown a significantly higher level of aromatic DNA adducts in normal adjacent breast tissue samples obtained from breast cancer patients than in those obtained from non-cancerous controls. The increased amount of DNA damage could be related to excess environmental carcinogen exposure and/or genetic susceptibility to such exposure. In the current study, we investigated the relationship between the levels of aromatic DNA adducts in breast tissues and polymorphisms of the drug-metabolizing genes cytochrome P4501A1 (CYP1A1), N-acetyltransferase-2 (NAT2), and glutathione S-transferase M1 (GSTM1), in 166 women having breast cancer. DNA adducts were measured using (32)P-postlabeling and information on smoking status was obtained from medical records. When pooled data of smokers and non-smokers were analyzed by multiple regression analyses, no significant correlation was found between the level of total DNA adducts and age, race, or polymorphisms of CYP1A1, GSTM1, and NAT2. The only significant predictor of the level of DNA adducts in breast tissues was smoking (P = 0.008). When data were analyzed separately in smokers and non-smokers, however, a significant gene-environment interaction was observed. Smokers with CYP1A1*1/*2 or *2/*2 genotypes had a significantly higher level of DNA adducts than those with the CYP1A1*1/*1 genotype. This effect was not seen among non-smokers. There was also a gene-gene interaction, as smokers with combined CYP1A1*1/*2 or CYP1A1*2/*2 genotypes and GSTM1 null had a much higher level of adducts than those with either CYP1A1 or GSTM1 polymorphism. Genetic polymorphisms of CYP1A1 and NAT2 were also significantly correlated with the frequency of certain types of DNA adducts. For example, a bulky benzo[a]pyrene (B[a]P)-like adduct was detected in 26% of the samples, the presence of which was not related to age, race, smoking status, or GSTM1 and NAT2 genotype. However, a significantly higher frequency of the B[a[P-like adduct was found in individuals having CYP1A1*1/*2 or *2/*2 genotypes than in those having the *1/*1 genotype (P = 0.04). In addition, individuals having slow NAT2 alleles had a significantly higher frequency of the typical smoking-related DNA adduct pattern, i.e. a diagonal radioactive zone (DRZ), than others did (P = 0.008). These findings suggest that polymorphisms of CYP1A1, GSTM1, and NAT2 significantly affect either the frequency or the level of DNA adducts in normal breast tissues of women having breast cancer, especially in smokers. Further large-scale studies are required to determine the exact role of these polymorphisms and types of DNA damage in breast cancer susceptibility.

The present research work was designed to study mussel's (Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to benzo[a]pyrene (B[a]P) exposure along with heat stress. Mussels were exposed to a sublethal dose of B[a]P [75 nM (19 μg/L/animal)] + temperature gradient (18, 20, 22, 24 and 26 °C) for 7 days. B[a]P hydroxylase (BPH) and glutathione-S-transferase (GST) activities were assessed in digestive gland tissues as phase I and phase II biotransformation parameters. Catalase (CAT) activity and malonedialdehyde (MDA) were measured as potential biomarkers of oxidative stress and lipid peroxidation. The cholinergic system was evaluated using acetylcholinesterase (AChE) activity. DNA damage was assessed using micronuclei (MN) test. BPH and GST activities showed a decreasing trend in B[a]P-exposed animals at 24 and 26 °C. CAT activity showed a bell-shaped response in B[a]P-exposed and in heat-stressed organisms at a maximum temperature of 22 °C. AChE activity was significantly inhibited in response to B[a]P being more pronounced at a temperature of 26 °C. MN in digestive gland cells suggest that B[a]P exposure induced significant DNA alteration with a maximum response in organisms coexposed to B[a]P and a temperature of 26 °C. Biomarker data are further discussed in relation B[a]P accumulation in mussels digestive gland. These data should be carefully considered in view of the biological effects of organic pollutants, particularly in organisms under the challenging effects of extreme temperature fluctuations.

Progressive decline in fertility and sperm quality has been reported over the last few decades, especially in industrialized nations. It has been proposed that exposure to factors that induce damage in DNA of spermatogenic cells may significantly contribute to impaired fertility. Here, the (32)P-postlabelling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 volunteers, either healthy subjects or patients with an impaired fertility. The levels of DNA adducts were 1.35-fold higher in the infertile group as compared to healthy individuals (P = 0.012). Similarly, a significant negative correlation between the levels of DNA adducts and measures of semen quality (sperm concentration and motility) has been observed (P <or= 0.001). In addition, the levels of bulky DNA adducts in sperm cells positively correlates with amounts of leukocytes in semen, which were significantly higher in semen of infertile subjects.

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

White blood cell DNA adducts were measured in coke oven workers, in residents from the area next to the coke oven in Silesia, Poland (highly industrialized region), and in residents from the rural area of Poland using the 32P-postlabeling technique. This method detected aromatic adducts including adducts formed by polycyclic aromatic hydrocarbons (PAHs). Highest levels of adducts in DNA were seen in the group of coke battery workers (6.9 adducts/10(8) nucleotides). Seasonal variations in levels of DNA adducts were observed both in residents of the district near the coke oven area and individuals from the rural area of Poland. Blood samples collected from people living near the coke oven in winter showed much higher levels of DNA adducts than blood samples obtained in summer (5.0 adducts/10(8) nucleotides in winter and 1.4 adducts/10(8) nucleotides in summer). The difference in the level of DNA adducts between winter and summer was smaller in the group of people living in the rural area (3.2 adducts/10(8) and 2.2 adducts/10(8), respectively). In most cases the levels of X-spots correlated with the levels of other DNA adducts. Correlation coefficients(r) between the levels of X-spots and other adducts ranged between 0.46 and 0.74 (p &lt; 0.05), except for coke oven workers where no correlation was observed.

White blood cell DNA adducts were measured in coke oven workers, in residents from the area next to the coke oven in Silesia, Poland (highly industrialized region), and in residents from the rural area of Poland using the 32P-postlabeling technique. This method detected aromatic adducts including adducts formed by polycyclic aromatic hydrocarbons (PAHs). Highest levels of adducts in DNA were seen in the group of coke battery workers (6.9 adducts/10(8) nucleotides). Seasonal variations in levels of DNA adducts were observed both in residents of the district near the coke oven area and individuals from the rural area of Poland. Blood samples collected from people living near the coke oven in winter showed much higher levels of DNA adducts than blood samples obtained in summer (5.0 adducts/10(8) nucleotides in winter and 1.4 adducts/10(8) nucleotides in summer). The difference in the level of DNA adducts between winter and summer was smaller in the group of people living in the rural area (3.2 adducts/10(8) and 2.2 adducts/10(8), respectively). In most cases the levels of X-spots correlated with the levels of other DNA adducts. Correlation coefficients(r) between the levels of X-spots and other adducts ranged between 0.46 and 0.74 (p < 0.05), except for coke oven workers where no correlation was observed.ImagesFIGURE 3.

DNA adduct formation among workers in a Thai industrial estate and nearby residents

Progressive decline in fertility and sperm quality has been reported over the last few decades, especially in industrialized nations. It has been proposed that exposure to factors that induce damage in DNA of spermatogenic cells may significantly contribute to impaired fertility. Here, the (32)P-postlabelling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 volunteers, either healthy subjects or patients with an impaired fertility. The levels of DNA adducts were 1.35-fold higher in the infertile group as compared to healthy individuals (P = 0.012). Similarly, a significant negative correlation between the levels of DNA adducts and measures of semen quality (sperm concentration and motility) has been observed (P

The rate of formation and the persistence of an exocyclic guanine adduct formed in DNA of rodents treated with various doses of N-nitrosopyrrolidine (NPYR) have been determined. NPYR is hepatocarcinogenic to the rat and forms a covalent adduct in liver DNA; this adduct was recently identified as 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2, 1-f]purine-4[3H]-one. Dose-dependent amounts of adduct formed in liver, kidney and lung DNA of rats, hamsters and mice given oral doses (56-900 mg/kg body wt) of NPYR. The persistence of the adduct in DNA after administration of low doses of NPYR to rats was greatest in the target organ, i.e. the liver; at high doses of NPYR, adduct levels in DNA changed little over a period of at least 72 h. In the hamster, in which NPYR is carcinogenic to the lung but apparently not the liver, the adduct level in liver DNA was an order of magnitude greater than in lung or kidney DNA for a dose of NPYR of 225 or 900 mg/kg body wt; persistence of the adduct in lung DNA was only slightly longer than in liver DNA. The formation and persistence of the 7,8-pyridoguanine adduct in the rat appeared to be consistent with the organotropy of this carcinogen, but this was not true for the hamster, a species that seems to be more resistant to induction of liver and kidney cancer by this carcinogen. Imidazole, an inhibitor of microsomal amine oxidase, and disulfiram, an inhibitor of aldehyde dehydrogenase, decreased metabolic activation of NPYR to an alkylating intermediate; inducers and inhibitors of cytochrome P450 monooxygenases had little effect on the metabolic activation of NPYR to an alkylating agent.

Bulky DNA adducts in human sperm: relationship with fertility, semen quality, smoking, and environmental factors

The importance of DNA adducts in carcinogenesis had been discussed. The 32P-postlabelling method was developed as a quantitative technique to measure the level of different DNA adducts including adducts in human DNA. The elevated level of DNA adducts was found in white blood cells in persons exposed environmentally and occupationally to high concentrations of PAHs (polycyclic aromatic hydrocarbons) in the ambient air. Tobacco also generated higher level of DNA adducts both in lymphocytes and laryngeal tissues of smokers. Exposure to styrene has been of interest world-wide because of the very high exposure and persistence of adducts in DNA of lamination workers.

Distribution of DNA adducts in the respiratory tract of rats exposed to diesel exhaust