Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N-dG) of the potent environmental carcinogen, benzo[a]pyrene

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question — what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? — has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N²-dG. In previous work, [+ta]-B[a]P-N²-dG was shown to be capable of inducing>95% G→T mutations in one sequence context (5′-T C), and 95% G→A mutations in another (5′-A A). This raises the question — how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N²-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G→T vs. G→A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N²-dG and AP-site mutagenesis, as well as the so-called “A-rule”.) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N²-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.     

Trace determination of urinary 3-hydroxybenzo[a]pyrene by automated column-switching high-performance liquid chromatography

3-Hydroxybenzo[a]pyrene (3-OHB[a]P), one of the metabolites of benzo[a]pyrene (B[a]P), has been determined in human urine using an automated column-switching procedure. The hydrolysed biological sample is centrifuged just prior to being injected into a reusable precolumn loop, which is packed with a preparative phase and coupled on-line to a liquid chromatographic (LC) system. A rapid pre-treatment of the hydrolysed sample, consisting of a concentration and a crude clean-up, is performed on the precolumn. The analytes are then non-selectively desorbed with the LC eluent and the sample is cleaned again in three successive purification columns using the direct transfer or “heart-cut” technique. The pre-treatment does not exceed 3 min. and the entire analytical purification and separation procedure takes less than 30 min. Average 3-OHB[a]P recovery reaches 95% in the 1–50 ng/l range of urine, and the detection limit is 0.1 ng/l urine for a 3 ml injection of hydrolysed urine. The developed method was compared with a more time-consuming off-line method to analyse urines of B[a]P gavaged rats; the statistical treatment indicates that both methods are in agreement. The method was applied to purify and concentrate the urine samples of workers exposed and apparently unexposed to polycyclic aromatic hydrocarbons (PAHs).     

Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: Removal of O-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 μmol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O⁶-methylguanine (O⁶-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P < 0.05), and colonic (at three sequential time points; ANOVA, F = 4.96, P < 0.05) O⁶-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P < 0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P = 0.0026, r = 0.83, r² = 0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

Inhibition of the benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 g ml^–1decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49–55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7–1.9 nmol min^–1 mg^–1 microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.     

Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease

Honey bees are important pollinators of commercial blueberries in the southeastern United States, and blueberry producers often use supplemental bees to achieve adequate fruit set. However, honey bees also vector the plant pathogenic fungus Monilinia vaccinii-corymbosi which infects open blueberry flowers through the gynoecial pathway causing mummy berry disease. Here, we report the results of a 3-year field study to test the hypothesis that using bee hives equipped with dispensers containing the biocontrol product Serenade, a commercial formulation of the bacterium Bacillus subtilis which has shown activity against flower infection by M. vaccinii-corymbosi in laboratory experiments, can reduce mummy berry disease incidence when honey bees are used as pollinators in blueberries. Individual honey bees carried 5.1–6.4 × 10⁵ colony-forming units (CFU) of B. subtilis when exiting hive-mounted dispensers with Serenade. On caged rabbiteye blueberry bushes in the field, population densities of B. subtilis vectored by honey bees reached a carrying capacity of <10³ CFU per flower stigma within 2 days of exposure, and there was a highly significant non-linear relationship between B. subtilis populations per stigma and bee activity, expressed as number of legitimate flower visits per time interval per cage (R = 0.6928, P < 0.0001, n = 32). Honey bee density (1600 or 6400 individuals per 5.8-m³ cage) and Serenade treatment (presence or absence of the product in hive-mounted dispensers) significantly (P < 0.05) affected the incidence of fruit mummification on caged bushes, whereby increasing bee density increased disease incidence and application of Serenade reduced disease levels. Taken together, results of this study suggest that use of a hive-dispersed biocontrol product such as Serenade as a supplement during pollination can reduce the risk of mummy berry disease. This may be a prudent practice that optimizes the benefits to pollination of high bee densities while reducing the associated disease-vectoring risk.     

P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats

The aim of this study was to investigate the synergistic hepatoprotective effect of lignans from Fructus Schisandrae chinensis (LFS) with Astragalus polysaccharides (APS) on chronic liver injury in male Sprague-Dawley rats. Subcutaneous injection of 10% CCl₄ twice a week for 3 months resulted in significantly (p<0.001) elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the liver, significantly elevated levels (p<0.001) of malondialdehyde (MDA), lowered levels of reduced glutathione (GSH) (p<0.05) and catalase (CAT) (p<0.001), superoxide dismutase (SOD) (p<0.01)were observed following CCl₄ administration. ‘LFS+ASP’ treatment of rats at doses of ‘LFS (45 mg/kg)+APS (150 mg/kg)’ and ‘LFS (135 mg/kg)+APS (450 mg/kg)’ displayed hepatoprotective and antioxidative effects than the administration of either LFS or APS, as evident by lower (p<0.005 or 0.001) levels of serum ALT, AST, ALP and hepatic MDA (p<0.001) concentration, as well as higher SOD (p<0.05 or 0.005), CAT activities(p<0.01 or 0.005), GSH concentration (p<0.05 or 0.005) compared to the toxin treated group. Histopathological examinations revealed severe fatty degeneration in the toxin group, and mild damage in groups treated with ‘LFS+APS’ were observed. The coefficients drug interaction (CDI) between each individual drug and their combination (at the same dose of their single treatment) of these foregoing parameters were all less than 1, indicating that LFS and APS display hepatoprotective and antioxidant properties and act in a synergistic manner in CCl₄ induced liver injury in rats.     

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

No carrier added synthesis of O-(2'-[18F]fluoroethyl)-L-tyrosine via a novel type of chiral enantiomerically pure precursor, NiII complex of a (S)-tyrosine Schiff base

O-(2′-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) has gained much attention as a promising amino acid radiotracer for tumor imaging with positron emission tomography (PET) due to favorable imaging characteristics and relatively long half-life of ¹⁸F (110 min) allowing remote-site application. Here we present a novel type of chiral enantiomerically pure labeling precursor for [¹⁸F]FET, based on NiII complex of a Schiff’s base of (S)-[N-2-(N′-benzylprolyl)amino]benzophenone (BPB) with alkylated (S)-tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH2CH2X (X = OTs (3a), OMs (3b) and OTf (3c)). A series of compounds 3a–c was synthesized in three steps from commercially available reagents. Non-radioactive FET as a reference was prepared from 3a in a form of (S)-isomer and (R,S) racemic mixture. Radiosynthesis comprised two steps: (1) n.c.a. nucleophilic fluorination of 3a–c (4.5–5.0 mg) in the presence of either Kryptofix 2.2.2.or tetrabutylammonium carbonate (TBAC) in MeCN at 80 °C for 5 min, followed by (2) removal of protective groups by treating with 0.5 M HCl (120 °C, 5 min). The major advantages of this procedure are retention of enantiomeric purity during the ¹⁸F-introduction step and easy simultaneous deprotection of amino and carboxy moieties in 3a–c. Radiochemically pure [¹⁸F]FET was isolated by semi-preparative HPLC (C18 μ-Bondapak, Waters) eluent aq 0.01 M CH₃COONH₄, pH 4/C₂H₅OH 90/10 (v/v). Overall synthesis time operated by Anatech RB 86 laboratory robot was 55 min. In a series of compounds 3a–c, tosyl derivative 3a provided highest radiochemical yield (40–45%, corrected for radioactive decay). Enantiomeric purity was 94–95% and 96–97%, correspondingly, for Kryptofix and TBAC assisted fluorinations. The suggested procedure involved minimal number of synthesis steps and suits perfectly for automation in the modern synthesis modules for PET radiopharmaceuticals. Preliminary biodistribution study in experimental model of turpentine-induced aseptic abscess and Glioma35 rat’s tumor (homografts) in Wistar rats has demonstrated the enhanced uptake of radiotracer in the tumor area with minimal accumulation in the inflamed tissues.     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Population genetic structure of striped mullet, Mugil cephalus, along the coast of China, inferred by AFLP fingerprinting

The striped mullet, Mugil cephalus, is an economically important species for both aquaculture and commercial fisheries in China. In this study, the amplified fragment length polymorphism (AFLP) technique was employed to analyze population genetic diversity and genetic distance between different populations with the aim of elucidating the population structure and gene flow of M. cephalus along the coast of China. A total of 230 fragments with 150–500 bp were identified from 118 individuals by five AFLP primer combinations. The polymorphic loci within populations varied from 46.52% to 64.78%, with an average of 53.91%, and the average heterozygosity from 0.1829 to 0.2282, with an average of 0.2074. The UPGMA phenograms of 118 individuals were constructed based on Dice similarity coefficients and four clusters were recognized. AMOVA analysis revealed that 60.7% of genetic variations were within populations and 39.3% between populations. The estimated genetic distance (φ_ST) value over all polymorphic loci across the six populations was 0.393 (p < 0.0010), indicating a strong population structuring. The pairwise φ_ST value ranged from 0.1112 to 0.5358, with an average of 0.3693. The population pairwise gene flows (average N_m = 0.73) are low. In addition, the result of the Mantel test showed that there was a significant correlation between geographic and genetic distances (r = 0.5434, p = 0.0050). It was speculated that there exist at least four distinct geographic populational subdivisions of M. cephalus along the Chinese coast. This research has provided new molecular data which will aid our understanding of the genetic structure of this species.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P–DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 μM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10⁶ bp versus 3.7 lesions/10⁶ bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3–10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mβ16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P–DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

Activation of genotoxins to DNA-damaging species in exfoliated breast milk cells

Exfoliated cells, isolated from breast milk samples donated by UK-resident women (n=15), were incubated, either immediately or after culture for 7 days, with one of a series of genotoxins, either in the presence or absence of the DNA-repair inhibitors, hydroxyurea (HU), and cytosine arabinoside (ara-C). The numbers of DNA single-strand breaks induced were then assessed as comet tail length (CTL) (μm) using the alkaline single cell–gel electrophoresis (‘Comet’) assay; cell viability was measured by trypan blue exclusion. The heterocyclic aromatic amines (HAAs) (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.4 mM), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) (1.67 mM), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) (1.77 mM)), a polycyclic aromatic hydrocarbon (benzo[a]pyrene (B[a]P) (0.36 mM)), a nitro-polycyclic aromatic hydrocarbon (1-nitropyrene (1-NP) (1.84 mM)) and aromatic amines (o-toluidine (0.85 mM), p-chloroaniline (0.71 mM)) each induced statistically significant (P<0.0001, Mann–Whitney test) increases in median CTLs in breast milk cells from all the donors examined when incubated (30 min, 37°C) in the presence of HU/ara-C. In some cases, these compounds were also active in the absence of the repair inhibitors. There were marked variations in comet formation between donors and between genotoxins. Cell culture appeared to increase the epithelial cell proportion and cultured cells retained their ability to activate genotoxins. The results suggest that breast milk is a valuable source of human mammary cells for the study of the metabolic activation of possible carcinogens.