Toxicity of aromatic hydrocarbons. VII. Hepatotoxicity of 9-nitrophenanthrene, and protection against it by beta-naphthoflavone

Male Sprague-Dawley rats were treated with a single i.p. injection of DMSO (3.0 ml/kg) or 9-nitrophenanthrene (9-NP, mg/kg) in DMSO. 9-NP produced a significant elevation of serum aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, and gamma-glutamyl transpeptidase (GGTP) levels relative to DMSO-injected rats 24 hr after injection. With the exception of GGTP, the increase in enzyme activities induced by 9-NP was significantly reduced by a 3-day pretreatment with beta-naphthoflavone (BNF; 40 mg/kg/day) in DMSO. The effect of 9-NP on GGTP levels was enhanced by BNF pretreatment.     

The therapeutic use of isometamidium chloride against Cryptobia salmositica in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss injected intramuscularly with isometamidium chloride (0.01 or 0.1 mg kg-1) at 3 wk post-infection and given a booster 2 wk later had significantly lower parasitaemias than infected controls. Packed cell volume increased after treatment and remained higher than in infected controls. The concentration of isometamidium in plasma was highest at 2 wk after injection and then declined. An intramuscular dose of 1.0 mg kg-1 of isometamidium chloride at 1, 2 and 3 wk postinfection (preclinical) significantly reduced the parasitaemia in rainbow trout 2 wk after treatment. A booster at 9 wk postinfection (chronic disease phase) reduced the parasitaemia further in all fish. The packed cell volume in these fish was higher than in infected controls. Treatment at 5, 6, and 7 wk postinfection (acute disease) had no effects and parasitaemias in treated fish were higher than in infected controls; also, anti-Cryptobia salmositica antibodies and titres of complement-fixing antibody were higher in these than in infected controls. Incubation of immune plasma or complement with isometamidium for 3 h did not affect the lytic titres of complement-fixing antibodies nor rainbow trout complement.     

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

Biological fate of [14C]-1-nitropyrene in rats following intragastric administration

1-Nitropyrene (1-NP), a weak carcinogen associated with diesel exhaust particles, has previously been detected in workplace atmospheres with in-use diesel engines and in the general environment. In order to gain insight in its biological fate, a single dose of [14C]-1-NP (27.6 microCi, 750 mg/kg body weight, b.w.) was administered intragastrically to rats and the presence of metabolites in blood and tissue homogenates, and radioactivity associated with blood proteins and tissue DNA, were studied. Early peak levels of radioactivity observed in blood and tissue homogenates indicated a rapid absorption of [14C]-1-NP from the gastrointestinal tract. Metabolite patterns observed in plasma, liver and kidney homogenates strongly suggested an important role of the intestinal microflora in the enterohepatic recirculation, but not in nitroreduction of 1-NP prior to absorption from the gastrointestinal tract. This might explain the low levels of radioactivity associated with blood proteins, since 1-nitrosopyrene, a product of nitroreduction of 1-NP, is likely to be involved in protein binding. Levels of radioactivity associated with plasma proteins were approximately four times higher than the levels of radioactivity associated with hemoglobin (401.0 and 84.1 pmol/g protein per micromol 1-NP kg b.w., respectively, at 24 h). Maximal 25% of the associated radioactivity was released following mild alkaline hydrolysis of either hemoglobin or plasma proteins. 1-Aminopyrene was the only released compound after hydrolysis of hemoglobin. In addition to 1-aminopyrene, two more polar unidentified metabolites were detected following hydrolysis of plasma proteins. Association of radioactivity with DNA was highest in the liver at the first moments of observation (7.4 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w.), but decreased rapidly to levels lower than observed for kidney DNA (max. 3.0 pmol 14C Eq./mg DNA per micromol 1-NP kg b.w. at 24 h). In lungs 8-50 times less radioactivity was associated with DNA than observed in the liver and kidneys. The results of this study show, that 1-NP undergoes an extensive and complex biotransformation in vivo, resulting in a variety of metabolites present in blood and tissue homogenates and a diversity of blood protein adducts. Concentrations of plasma metabolites, blood protein adducts and DNA adducts were rather low. In addition, previous studies also showed relatively low concentrations of metabolites present in urine. Therefore, sensitive and selective methods will be needed in order to evaluate the biological fate of 1-NP, associated with diesel exhaust particles, in humans.     

The influence of piscivory on life history traits of brown trout

Brown trout in Lake Femund migrated from the nursery streams mainly at 2 years old, but ranging between 1 and 8 years. Brown trout switched to piscivory from 3 years onwards, and a body length of 17·5 cm, according to back calculation from scales. Fast growers switched to piscivory at a younger age and smaller size than slow growers. The most slow-growing trout switched to fish feeding at 9 years old and a mean body length of 36 cm. The growth of the invertebrate feeders was almost rectilinear to c. 45 cm and 11 years of age. Switching to a fish diet induced increased growth rates. Age at sexual maturity increased with the age at which the fish became piscivorous. The invertebrate feeders matured at an age similar to that of the most fast growing piscivorous trout. The mortality rate of sub-adults and adults did not differ significantly between fish and invertebrate feeding trout. Longevity of piscivorous trout was estimated at 11 years and of invertebrate feeders at 10 years.     

Nitropyrenes are inducers of polyoma viral DNA synthesis

The biological activity of a series of nitropyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a ts-a mutant of polyoma virus. Concentrations of 10-30 micrograms/ml of 1-nitropyrene (1-NP) induced viral replication, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 micrograms/ml) to the culture medium. The response was less than that obtained with 0.1 micrograms/ml of the activated metabolite of benzo[a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replication, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5-2.0 microgram/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 microgram/ml indicated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modification seen with 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.     

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence. A slipped frameshift intermediate model for dinucleotide deletion

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

Haemolytic activity in the serum of brown trout, Salmo trutta

Brown trout serum contains a natural, spontaneous, antibody-independent lytic activity and a haemolysin antibody complement-mediated lytic activity against unsensitized and trout antibody-sensitized sheep erythrocytes, respectively. The use of various activators and inactivators of the mammalian complement system demonstrated that trout serum possesses complement or complement-like components similar in activity to those present in the classical and alternative pathways found in mammals. A single injection of trout with sheep erythrocytes stimulated the production of antibody-secreting cells in lymphoid organs and increased the levels of natural haemolysins. A second injection of sheep erythrocytes further raised the haemolysin values and antibody-secreting cell counts. Serum complement from homologous or closely related fish species was more effective for use in the haemolysin and antibody-secreting cell assays than that from heterologous sources, except guinea pig. Based on physico-chemical properties, gel filtration and immunoelectrophoretic studies, natural and induced anti-sheep erythrocyte haemolysins were found to be similar molecules and are possibly high molecular weight IgM antibodies.     

Formation of benzo[a]pyrene-DNA adducts by microsomal enzymes: comparison of maternal and fetal liver, fetal hematopoietic cells and placenta

The formation of benzo[a]pyrene (BP)-DNA adducts was studied in vitro in the presence of microsomes prepared from the isolated labyrinth zone of the rat placenta, the hematopoietic erythroblast cells of the fetal liver, the fetal liver, as well as the maternal liver. Pregnant rats received beta-naphthoflavone (beta NF; 15 mg/kg, i.p.) on day 17 gestation. One day later, placentae, fetal and maternal livers were obtained and hematopoietic erythroblast cells were separated from hepatocytes in the fetal livers. The respective microsomal fractions were incubated in the presence of calf thymus DNA, NADPH-regenerating system and [3H]BP (300 microCi) at 37 degrees C for 30 min. Following beta NF pretreatment, the levels of covalent binding (pmol/mg DNA/mg microsomal protein) for maternal liver, fetal liver, placenta and erythroblast cells were: 28.4, 2.4, 0.31 and 3.9, respectively, with the hematopoietic erythroblast cells being the most active among fetal tissue preparations. The extent of transplacental induction compared to control was greatest in the hematopoietic cells (18-fold) followed by fetal liver (16-fold) and labyrinth zone (5-fold). Further experiments characterized the BP-DNA adducts formed by induced microsomes. DNA was isolated, purified and digested sequentially with DNase I, snake venom phosphodiesterase type II and alkaline phosphatase type III. The deoxynucleoside-BP adducts were purified on a Sephadex LH-20 column and then separated on HPLC and the adducts were quantitated radiometrically. Seven distinct adducts were separated on HPLC and named A-G in order of elution. Adduct B was prominent in all preparations (22-55% total radioactivity). The adduct profile and retention time for peak B is similar to that reported for the adduct formed by microsomal activation of 9-hydroxy BP. Peak D constituted a major fraction (19%) in maternal liver profiles in comparison with the three fetal tissue preparations (8%). In subsequent experiments, peak D was shown to be derived from reaction of (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) with DNA. Peak C was unique to erythroblast cell and labyrinth profiles, while peak G was specific for maternal liver and fetal liver profiles. These results demonstrate that fetal liver and its hematopoietic cells are significant sites of BP bioactivation which may contribute to the fetal toxicity of polyaromatic hydrocarbons.     

Heart rate responses to predation risk in Salmo trutta are affected by the rearing environment

Both wild‐ and hatchery‐reared brown trout Salmo trutta , 18 months of age and of the same genetic origin, responded with increased heart rates (tachycardia) to a simulated predator attack on 2 consecutive days. Brown trout reared in the hatchery showed a more rapidly induced tachycardia compared with wild‐reared fish at day 1, but not day 2. During an undisturbed period several hours after attacks, hatchery‐reared brown trout maintained higher heart rates compared to wild‐reared fish on both days. Behavioural responses to the attack were very low for all fish, although hatchery‐reared fish tended to be more active than wild fish after the attack day 2. The observed differences may have had a genetic background caused by different selection regimes in the hatchery‐ and wild‐rearing environments, or could have been due to different phenotypic responses in the two environments.     

Intracellular localization of hsp90 is influenced by developmental stage and environmental estrogens in rainbow trout Oncorhynchus mykiss

In this study, we investigated the intracellular localization of heat shock proteins hsp90 and hsp70 in adult and juvenile rainbow trout (Oncorhynchus mykiss) and in juvenile trout exposed to estrogen or one of its mimics, 4-nonylphenol (4-NP). Livers were harvested from each group and analyzed directly or separated into nuclear and nonnuclear fractions. We found that hsp70 was predominantly nonnuclear in mature and juvenile fish regardless of treatment. Mature fish had significantly greater levels of hsp90 outside the nucleus, while juvenile fish had similar levels of hsp90 inside and outside the nucleus. Treatment with estradiol or 4-NP resulted in a translocation of hsp90 out of the nucleus in juvenile fish. To our knowledge, this is the first study to demonstrate a development- and/or estrogen-dependent shift in intracellular localization of hsp90 in fish. This change in subcellular distribution points to important roles for this hsp in fish estrogen signaling and development.     

Effect of exogenous and endogenous antioxidants on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions: insights into Huntington's disease

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of complex II in the mitochondria. 3-NP toxicity has gained acceptance as an animal model of Huntington's disease (HD). In the present study, we confirmed that rats injected with 3-NP (20 mg/kg, i.p., daily for 4 days) exhibit increased oxidative stress in both striatum and cortical synaptosomes as well as lesions in the striatum. Synaptosomal membrane proteins from rats injected with 3-NP exhibited a decrease in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation, and western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal protein oxidation. Treatment of rats with the brain-accessible free radical spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO; 30 mg/kg, i.p., daily 2 h before 3-NP injection) or with N-acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3-NP injection), a known glutathione precursor, before 3-NP treatments protects against oxidative damage induced by 3-NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3-NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3-NP neurotoxicity and perhaps against HD as well.     

Carcinogen aflatoxin B1 is located preferentially in internucleosomal deoxyribonucleic acid following exposure in vivo in rainbow trout

The purpose of this work was to investigate the distribution in chromatin of deoxyribonucleic acid (DNA) adducts of aflatoxin B1, following exposure in vivo. Rainbow trout were injected intraperitoneally with radiolabeled aflatoxin B1, a potent procarcinogen known to readily induced hepatocellular carcinomas in these fish. After maximum incorporation, liver nuclei were prepared and digested with micrococcal nuclease. Mono-, di-, and trinucleosomal fractions were purified from several stages of nuclease digestion, and the lengths and specific activities of their DNA were determined. The results indicate that aflatoxin B1 is approximately 5 times as likely on a per nucleotide basis to localize on internucleosomal (linker) DNA as on nucleosomal core DNA in this system.     

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.     

Binding of benzo[a]pyrene to DNA by cytochrome P-450 catalyzed one-electron oxidation in rat liver microsomes and nuclei

To investigate whether cytochrome P-450 catalyzes the covalent binding of substrates to DNA by one-electron oxidation, the ability of both uninduced and 3-methylcholanthrene (MC) induced rat liver microsomes and nuclei to catalyze covalent binding of benzo[a]pyrene (BP) to DNA and formation of the labile adduct 7-(benzo[a]pyren-6-yl)guanine (BP-N7Gua) was investigated. This adduct arises from the reaction of the BP radical cation at C-6 with the nucleophilic N-7 of the guanine moiety. In the various systems studied, 1-9 times more BP-N7Gua adduct was isolated than the total amount of stable BP adducts in the DNA. The specific cytochrome P-450 inhibitor 2-[(4,6-dichloro-o-biphenyl)oxy]ethylamine hydrobromide (DPEA) reduced or eliminated BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The effects of the antioxidants cysteine, glutathione, and p-methoxythiophenol were also investigated. Although cysteine had no effect on the microsome-catalyzed processes, glutathione and p-methoxythiophenol inhibited BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The decreased levels of binding of BP to DNA in the presence of glutathione or p-methoxythiophenol are matched by decreased amounts of BP-N7Gua adduct and of stable BP-DNA adducts detected by the 32P-postlabeling technique. This study represents the first demonstration of cytochrome P-450 mediating covalent binding of substrates to DNA via one-electron oxidation and suggests that this enzyme can catalyze peroxidase-type electron-transfer reactions.     

Determination of genotoxicity of the metabolites of the pesticides Guthion,Sencor, Lorox,Reglone, Daconil and Admire by 32P-postlabeling

Commercial formulations of the pesticides: Guthion (azinphos methyl), Sencor (metribuzin), Lorox (linuron), Reglone (diquat), Daconil (chlorothalonil) and Admire (imidacloprid) were studied for their genotoxicity by 32P-postlabeling. Metabolites of the pesticides were obtained enzymatically using arochlor induced rat liver S9 fraction, in an NADPH generating system. The resulting metabolites were reacted with calf thymus DNA and the DNA was analyzed for presence of adducts by either the nuclease P1 or butanol enrichment. Nuclease P1 enrichment resulted in adducts for all the pesticides. Compared to the level of adducts in control DNA, the levels in pesticide-treated DNA were higher for all the pesticides, except Daconil. The increase in adduct numbers for pesticide-treated DNAs ranged from 4.9-12.4 times the control-DNA indicating pesticide genotoxicity in this in vitro system. Enrichment using butanol extraction gave three adducts unique to Sencor-DNA. These adducts were different from those obtained with nuclease P1 enrichment of the same. B()P was the positive control for the in vitro metabolism, and two adduct enrichment procedures: nuclease P1 digestion and butanol extraction.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.