Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.     

Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants — 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite — and some herbicides (lindane and the isomers α, β, δ; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.     

Integrated use of biomarkers and bioaccumulation data in Zebra mussel (Dreissena polymorpha) for site-specific quality assessment.

One of the useful biological tools for environmental management is the measurement of biomarkers whose changes are related to the exposure to chemicals or environmental stress. Since these responses might vary with different contaminants or depending on the pollutant concentration reached in the organism, the support of bioaccumulation data is needed to prevent false conclusions. In this study, several persistent organic pollutants -- 23 polychlorinated biphenyl (PCB) congeners, 11 polycyclic aromatic hydrocarbons (PAHs), six dichlorodiphenyltricholroethane (DDT) relatives, hexachlorobenzene (HCB), chlorpyrifos and its oxidized metabolite -- and some herbicides (lindane and the isomers alpha, beta, delta; terbutilazine; alachlor; metolachlor) were measured in the soft tissues of the freshwater mollusc Zebra mussel (Dreissena polymorpha) from 25 sampling sites in the Italian portions of the sub-alpine great lakes along with the measure of ethoxyresorufin dealkylation (EROD) and acetylcholinesterase (AChE) activity. The linkage between bioaccumulation and biomarker data allowed us to create site-specific environmental quality indexes towards man-made chemicals. This classification highlighted three different degrees of xenobiotic contamination of the Italian sub-alpine great lakes: a high water quality in Lake Lugano with negligible pollutant levels and no effects on enzyme activities, an homogeneous poor quality for Lakes Garda, Iseo and Como, and the presence of some xenobiotic point-sources in Lake Maggiore, whose ecological status could be jeopardized, also due to the heavy DDT contamination revealed since 1996.     

DNA strand breaks and adducts determined in feral and caged chub (Leuciscus cephalus) exposed to rivers exhibiting variable water quality around Birmingham, UK

This study forms part of an investigation into the effects on fish of immersion in three rivers around Birmingham, UK. The rivers Blythe, Cole and Tame exhibit relatively high, intermediate and poor overall water quality, respectively, according to combined levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), as well as heavy metals. Specifically, biomarkers of genotoxicity (DNA strand breaks and adducts) were measured in feral and caged chub (Leuciscus cephalus), complementing another study in which data were presented for a number of other hepatic biomarkers measured in the same animals. In both feral and caged chub, there was a general elevation of DNA strand breaks with a decrease in chemical water quality, with some time points exhibiting significantly higher levels at the most (Tame) compared with least polluted sites (Blythe), particularly in the cage-held animals. Combined-season DNA adduct data suggested a higher degree of toxic insult in the feral compared with caged chub and revealed particularly high levels of adducts in fish caught from the Cole. The pattern of adducts shown was typical of exposure to a complex mixture of PAHs which were relatively high, and similar, in both the Cole and Tame. Overall, these data are consistent with exposure of both feral and caged chub to contaminants which are able to induce specific, moderately genotoxic effects.     

Interspecies differences in DNA single strand breaks caused by benzo(a)pyrene and marine environment

The presence of DNA single strand breaks in untreated specimens of selected species, mosquito fish Gambusia affinis, painted comber Serranus scriba, blue mussel Mytilus galloprovincialis, spiny crab Maja crispata and sea cucumber Holothuria tubulosa as well as in 10 microg/g benzo(a)pyrene (BaP) treated mosquito fish, blue mussel and spiny crab was measured, using alkaline filter elution. Interspecies differences in alkaline elution profiles were observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. Spiny crab hemocytes are more sensitive to action of BaP then blue mussel hemocytes and mosquito fish hepatocytes that could be explained by differences in the rates of distinct metabolic reactions and DNA repair among the investigated species. In field study, DNA single strand breaks were measured in hepatocytes of painted comber and in hemocytes of blue mussel and spiny crab from natural population specimens collected at eight sampling sites along Istrian coast, Croatia. Spatial variations in DNA integrity for each species were detected and revealed for the first time that spiny crab is responsive to different environmental conditions. Interspecies variations in the DNA integrity due to environmental conditions, confirmed species specific susceptibility to genotoxicity of certain environment that in long-term may modify the structure of marine communities. The multi-species approach in designing biomonitoring studies was suggested.     

Quantitation of chemically induced DNA strand breaks in human cells via an alkaline unwinding assay

DNA strand breaks induced in human CCRF-CEM cells by electrophilic chemicals (carcinogens/mutagens) can be readily quantitated via a facile alkaline unwinding assay. This procedure estimates the number of chemically induced DNA strand breaks on the basis of the percentage DNA converted from double-stranded to single-stranded form during an exposure to the alkaline unwinding conditions. The assay is based on the assumption that each strand break serves as a strand unwinding point during the alkaline denaturation. The extent of strand separation can be standardized with respect to the initial level of induced strand breaks by the use of X-rays, which produce known levels of DNA strand breaks per rad in mammalian cells. Subsequent to the alkaline exposure, the single- and double-stranded DNA were separated by use of thermostated hydroxylapatite columns (60 degrees C), and the DNA was quantitated via a fluorescence assay (Hoechst 33258 compound). A correlation was shown between mammalian DNA strand-breaking potential (as measured in this procedure) and the propensity of these chemicals to revert Salmonella typhimurium TA100.     

Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.     

Multi-biomarker responses after exposure to organophosphates chlorpyrifos in the freshwater mussels Unio tigridis and snails Viviparous benglensis

Effects of chlorpyrifos (CPF) on soft tissues of mollusks, Unio tigridis and Viviparous benglensis, have not been studied for biochemical and molecular traits. Therefore, the changes of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), malondialdehyde (MDA), acetylcholinesterase (AChE), and DNA damage in these mollusks were assessed during 21-day exposure to three concentrations of CPF. The results showed that SOD and CAT enzymes in the two mollusk species decreased with highest CPF concentration, while MDA significantly increased with highest CPF concentration. AChE concentrations also showed a clear reduction in soft tissues of both mollusks. DNA damage parameters showed significant differences between controls and three CPF concentrations for each parameter measured in the digestive gland of U. tigridis; and for V. bengalensis significant differences were observed between controls and highest two concentrations of CPF for comet length and tail length. CPF has the ability to prevent reproduction status for the snail V. bengalensis compared to the controls. In this study, we can conclude from the significant changes occurring in the concentrations of CAT, SOD, MDA, AChE, and DNA damage that the concentrations of these biomolecules in the soft tissues of the mussel and snail can be considered suitable biomarkers for a sublethal exposure and/or effects of CPF at the tested concentrations, and we can then use it in biomonitoring of water bodies.     

Acetylcholinesterase and ethoxyresorufin-o-deethylase in the surgeonfish Acanthurus bahianus around Martinique Island (French West Indies)

Abstract

The biochemical indicators of biological effects of pollutants, ethoxyresorufin-edeethylase (EROD) and acetylcholinesterase (AChE), were measured in Acanthurus bahianus from 10 stations around Martinique Island (French West Indies). Strong induction of EROD was demonstrated in the bay of Fort de France in relation to organic pollution. Depression of AChE may suggest that neurotoxic compounds are having some effects along the east coast.     

Biomarker-enhanced assessment of reproductive disorders in Monoporeia affinis exposed to contaminated sediment in the Baltic Sea

Introducing biomarkers into monitoring programs requires understanding of their responses in relation to higher-level biological effects as well as modulating effects of confounding environmental factors. We evaluated relationships between the general toxicity biomarkers (acetylcholinesterase [AChE], lysosomal membrane stability [LMS], oxygen radical absorbance capacity [ORAC]) and reproductive performance (fecundity and embryo aberrations) in the amphipod Monoporeia affinis in the Baltic Sea. To further link biomarker response to contaminant (PCBs, PAHs and metals) levels in the surrounding sediments as well as environmental factors (salinity, bottom depth and total organic carbon in sediments [TOC]), correlation and partial least square regression (PLSR) analyses were applied. The observed contaminants levels were frequently elevated for heavy metals and PAHs, but not PCBs. In the amphipod populations, female ORAC values were positively related to the occurrence of females carrying malformed or membrane-damaged embryos and to the percentage of such embryos in their broods, but also to the fecundity. Female AChE activity was negatively related to the frequency of the membrane-damaged embryos, and positively to the frequency of embryos with arrested development in the broods. Moreover, higher AChE activity and ORAC values in the females occurred at elevated concentrations of metals and PAHs, while there was a negative correlation between embryo ORAC and some PCB congeners. The PLSR models explained over 80% of the variation in the female ORAC and AChE values by variation in contaminant concentrations in combination with environmental variables. Specifically, CB180 and PAM4,9 were identified as negative predictors for ORAC, whereas many PAHs and some metals were positive predictors. The AChE activity was positively related to some metals and negatively to PCBs. In the PLSR models, environmental factors had significant modulating effects, with positive effect of salinity on female ORAC and AChE, and negative effect of TOC on the AChE. The LMS data were less informative, with no apparent relation to any of the contaminants. Linking subcellular responses to the reproduction effects facilitates environmental stress assessment and understanding of the response mechanisms, but also calls for more experimental and field data providing a mechanistic understanding to these linkages.     

Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels

A biomonitoring programme of wild and caged mussels (Mytilus galloprovincialis) was carried out at four selected sites along the Ligurian coast: Cornigliano, Voltri, Zinola, and Sanremo (Italy). Mussels of a very narrow size range were left in situ for 30 days. Adult specimen of mussels from natural substrates were collected in the same areas. Animals from a mussel farm located in La Spezia were used as controls. Micronucleus frequency and DNA single strand breaks, evaluated by alkaline elution, were used as biomarkers of genotoxicity. Mussels were also analyzed for polycyclic aromatic hydrocarbons (PAH) and heavy metals (Hg and Cd). Different gradients of PAH and metal concentrations were detected in tissues of mussels from different samplings sites. A weak correlation was found between single strand breaks and PAH content while MN frequency correlated with Hg concentration (r = 0.28, P < 0.002). A clear distinction between the sites, allowing classification along a pollution gradient (Sanremo < Zinola < Voltri < Cornigliano) was demonstrated by the analysis of genotoxicity parameters. The obtained results suggested that the micronucleus assay compared with DNA damage determination by alkaline elution allow to better discriminate the selected sites. DNA damage expressed as constant of elution (k ml(-1) x 10(3)) ranges from 30 +/- 9.6 to 89.60 +/- 40.10, and micronuclei frequency from 1.78 +/- 1.04 to 24.4 +/- 12.9, in control animals and in mussels from the most polluted site, respectively. Wild mussels accumulated significant concentrations of chemicals and showed a higher induction of chromosomal damage than caged mussels, expressed as micronuclei frequency. Caged mussels showed higher level of DNA damage than wild mussels, probably as a result of recent exposure. DNA damage was higher in September than in May, as opposed to micronuclei frequency being higher in May than in September. Endogenous and exogenous factors, such as change of pollutant input levels or compositions, could be considered the cause of such variability.     

Benzo(a)pyrene-induced genotoxicity: Attenuation by farnesol in a mouse model

In the present study we have evaluated the antigenotoxic effects of Farnesol (FL) a 15-carbon isoprenoid alcohol against benzo (a) pyrene [B(a)P] (125 mg kg^{? 1}.b.wt oral) induced toxicity. B(a)P administration lead to significant induction in Cytochrome P450 (CYP) content and aryl hydrocarbon hydrolase (AHH) activity (p < 0.001), DNA strand breaks and DNA adducts (p < 0.001) formation. FL was shown to suppress the activities of both CYP and AHH (p < 0.005) in modulator groups. FL pretreatment significantly (p < 0.001) restored depleted levels of reduced glutathione (GSH), quinone reductase (QR) and glutathione –S-transferase (GST). A simultaneous significant and at both the doses reduction was seen in DNA strand breaks and in in-vivo DNA adducts formation (p < 0.005), which gives some insight on restoration of DNA integrity. The results support the protective nature of FL. Hence present data supports FL as a future drug to preclude B (a) P induced toxicity.     

Potential health effects of exposure to carcinogenic compounds in incense smoke in temple workers

Incense smoke is a potential hazard to human health due to various airborne carcinogens emitted from incense burning. This study aimed to evaluate the potential health effects of exposure to benzene, 1,3-butadiene, and polycyclic aromatic hydrocarbons (PAHs) emitted from incense smoke in temple workers. Exposure and health risks were assessed through the measurement of ambient exposure as well as through the use of biomarkers of exposure and early biological effects. Ambient air measurement showed that incense burning generates significantly higher levels of airborne benzene (P<0.01), 1,3-butadiene (P<0.001) and total PAHs (P<0.01) inside the temples, compared to those of the control workplace. Temple workers were exposed to relatively high levels of benzene (45.90 microg/m(3)) 1,3-butadiene (11.29 microg/m(3)) and PAHs (19.56 ng/m(3)), which were significantly higher than those of control workers (P<0.001). The most abundant PAHs were chrysene, B[ghi]P, B[a]P, B[a]F and fluoranthene. Concentrations of B[a]P and B[a]P equivalents in air samples to which temple workers were exposed were 63- and 16-fold, higher, respectively, than those to which control subjects were exposed (P<0.001). Biomarkers of exposure to benzene (blood benzene and the urinary metabolites trans,trans-muconic acid and S-phenylmercapturic acid), 1,3-butadiene (urinary monohydroxy-butenyl mercapturic acid) and PAHs (1-hydroxypyrene) were all significantly higher in temple workers than those in control workers. DNA damage and DNA repair capacity were measured as biomarkers of early biological effects. Temple workers had a significant increase in DNA damage observed as a 2-fold increase in the levels of leukocyte 8-hydroxy-2'-deoxguanosine (8-OHdG) and DNA strand breaks (P<0.001). A significant reduction of DNA repair capacity in temple workers determined by the radiation challenge assay was also observed. These results indicate that exposure to carcinogens emitted from incense burning may increase health risk for the development of cancer in temple workers.     

Recovery of Historically Contaminated Watercourse Polluted by the Chemical Wood Industry: EROD Activity in Fish as Biomarker

Despite outstanding process alterations over decades, pulp- and paper-mill-contaminated sediments and continuing exposure by the effluents may still have effects on biota. In this study, ecotoxicological impacts in the boreal watercourse were analyzed by measuring ethoxyresorufin-O-deethylase (EROD) induction from wild fish populations and from experimentally exposed fish. In order to assess the role of sediment-borne chemicals, juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to the surface sediments of Lake Vatianjärvi and Southern Lake Saimaa, both watercourses impacted by the chemical wood industry for approximately a century. Hepatic EROD activity was also measured from roach (Rutilus rutilus) and perch (Perca fluviatilis) caught in Lake Vatianjärvi. Increased EROD activity was not observed in wild fish caught in Lake Vatianjärvi nor in rainbow trout exposed to the sediment of Lake Vatianjärvi, but it was observed in rainbow trout exposed to the sediment of Southern Lake Saimaa, probably explained by critically high concentrations of retene. The results of both the laboratory and field study indicate the absence of EROD-inducing compounds of the previously heavily contaminated Lake Vatianjärvi.     

Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds

An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure.Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).     

Embryolethality and induction of 7-ethoxyresorufin O-deethylase in chick embryos by polychlorinated biphenyls and polycyclic aromatic hydrocarbons having Ah receptor affinity.

The lethality and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency of some individual polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs) in chick embryos were measured in order to compare the mechanisms of action of these compounds. In previous studies it was found that coplanar PCBs and certain PAHs have a high embryolethality in the chicken and that they induce embryonic EROD activity. Although the most potent PAHs were almost as embryolethal as the PCBs when injected into hens' eggs 72 h prior to measurement, they were considerably less potent EROD inducers. In the present study, three coplanar PCBs (3,3',4,4'-tetrachlorobiphenyl (TCB), 3,3',4,4',5-pentachlorobiphenyl (PeCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (HCB)) and four of the most toxic PAHs (benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), indeno[1,2,3-cd]pyrene (IP) and dibenzo[a, h]-anthracene (DBahA] were administered to chick embryos in different ways, including co-administration. Additive embryolethality was found when BkF and PeCB were co-administered as well as when BaA and DBahA were given simultaneously. The PAHs were more effective as EROD inducers when injected on day 9 (24 h prior to measurement) than when injected on day 7 (72 h prior to measurement). The opposite was found for PeCB and HCB, whereas no difference in potency was noted when comparing TCB injected 24 and 72 h before EROD determination. These substance-related differences were probably due, at least partly, to differences in biotransformation rates. EROD activities found after treatment with high doses of BkF, IP, or DBahA on day 9 were similar to those measured after treatment with PeCB in doses high enough to give maximal induction. Co-administration of high doses of BkF and PeCB did not further increase the activity, indicating that the PAHs and coplanar PCBs induce EROD to a common maximal value. To decrease the influence of metabolization of the PAHs on their EROD-inducing potency, EROD was determined early in development (day 8) and soon after treatment (24 h) in one experiment. In that experiment, the PAHs proved to be only a few times less potent EROD inducers in relation to their embryolethalities compared with the PCBs. The results of the present study, a previously observed similarity in pathology between chick embryos treated with PAHs and embryos treated with coplanar PCBs, and the fact that the most toxic PAHs also are the most avid Ah receptor binders suggest that the coplanar PCBs and the PAHs largely exert their toxicity in chick embryos via an Ah receptor-mediated mechanism. The differences between the compounds in their EROD-inducing potency/embryolethality ratios could probably be explained by their different rates of biotransformation.     

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.     

Alterations in DNA metabolism in Elliptio complanata mussels after exposure to municipal effluents

This study sought to examine the genotoxic potential in Elliptio complanata freshwater mussels exposed to a physically and chemically treated municipal effluent before and after ozone treatment. Mussels were continuously exposed to increasing concentrations of the effluents for 14 days. Genotoxicity was determined by tracking changes in key enzymes for purine and pyrimidine synthesis (dehydrofolate reductase and aspartate transcarbamoylase), catabolism of purines (xanthine oxido-reductase) and DNA strand-break levels as determined by the alkaline precipitation assay. Other biomarkers related to xenobiotic biotransformation (cytochrome P4503A and glutathione S-transferase activities), metal metabolism (labile zinc and redox state of metathioneins) and oxidative stress (superoxide dismutase activity) were also determined in the mussels. The data revealed that dehydrofolate reductase activity was reduced by the initial effluent and increased by the ozonated effluent. Aspartate transcarbamoylase activity was significantly induced only with the ozonated effluent. The levels of DNA strand breaks responded in a biphasic manner in mussels exposed to the physically and chemically treated effluent where an initial decrease was observed at a low effluent concentration (3% v/v) followed by an increase in DNA strand breaks at a higher effluent concentration (20%). This response pattern was lost in the ozonated effluent, where only a decrease in DNA breaks was found. Xanthine oxidoreductase activity was not significantly affected but did correlate significantly with dehydrofolate reductase activity. Multivariate factorial and canonical analyses revealed that oxidative stress and metal/xenobiotic metabolism markers were strongly correlated with DNA strand breaks in mussels, suggesting that the presence of metals (zinc) and planar hydroxylated hydrocarbons present in these effluents were strong contributors to the observed response. We conclude that municipal effluents contain a complex mixture of pollutants that could modulate DNA synthesis and repair mechanisms in mussels.     

Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10⁵], and this precision is improved to about [1/10⁷] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G^O) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G^O/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G^O might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)