Factors Influencing the Photodynamic Action of Benzo[a]pyrene on Escherichia coli

Death of Escherichia coli resulted when a buffer suspension was exposed simultaneously to colloidal benzo[a]pyrene (BP) and 355-mmu illumination. Neither hydrocarbon nor illumination alone caused death; oxygen had to be present. The survival curve had a shoulder, and then death proceeded exponentially with time. Death rate was independent of temperature between 6 and 32 C. The duration of the shoulder, however, decreased slightly with increase in temperature. The shoulder was not due to delay in BP entering the cell. Death was influenced by the composition of the medium in which the cells were grown prior to illumination. The amount of BP bound to the cells was determined after three ethyl alcoholether extractions. Appreciable binding occurred in the presence of 355-mmu illumination with air, and relatively little binding occurred under nitrogen; very little binding occurred in the dark with nitrogen or air. At the outset, rate of binding under illumination with air was not temperature-dependent, but with time it became strongly temperature-dependent. Binding under illumination with nitrogen was temperature-independent. Bound BP was associated primarily with cell protein. Cells in growth medium resisted death and BP binding. At 21 and 32 C, deoxyribonucleic acid damage occurred during exponential death. No damage was detected at 21 and 32 C in the dark with BP, under illumination in absence of BP, or under illumination with BP in a nitrogen atmosphere.     

Construction of a umuDC operon substitution mutation in Escherichia coli.

Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene. The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.     

Suppressor genes of a dnaA temperature sensitive mutation in Escherichia coli

Summary Recombinant plasmids were constructed from EcoRI digests of Escherichia coli chromosomal DNA and pMB9 DNA by selecting for suppression of a dnaA–T46 temperature-sensitive mutation. Two types of plasmid capable of suppressing the dnaA mutation were isolated. They did not carry any genetic markers around dnaA and physical mapping with various restriction enzymes showed that neither of the plasmids contained the dnaA gene. One plasmid, pYT47, was characterized further and the protein responsible for the suppression was identified by two-dimensional gel electrophoresis. The molecular weight of the suppressor protein was about 68 Kdal and thus is clearly different from the dnaA gene product.     

Benzo(a)pyrene hydroxylase activity of immunocompetent cells

A study was made of aryl hydrocarbon hydroxylase activity in immunocompetent cells of varying origin and in hepatocytes from CBA mice. The cells from intact animals may be arranged in the following way with regard to the activity of the enzyme: macrophages greater than hepatocytes much greater than thymocytes greater than splenocytes. The immunostimulants (tilorone and its analogs) altered benzo(a)pyrene hydroxylase activity depending on the cell type.     

Benzo(a) pyrene metabolism in vitamin A deficient rats

Hepatic microsomal metabolism of benzo(a)pyrene, a representative carcinogenic polycyclic hydrocarbon and an ubiquitous environmental pollutant was studied in control and vitamin A deprived (10–12 weeks) male rats. Hydroxylation of benzo(a)pyrene to fluorescent phenols was found to be significantly depressed in the deficient animals. The decreased hepatic metabolism may lead to delayed clearance of the carcinogenic chemicals in this condition and thus may explain at least in part the enhanced susceptibility to carcinogenesis in hypovitaminosis A.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^?3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way.

The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and gamma-radiation. The induction levels of polB::lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, H2O2 or ethanol. Pentachlorophenol did not significantly induce any din genes. gamma-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ. Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD. We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner. The polB gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and H2O2 or nalidixic acid and ethanol. In addition, dinB and dinD in the presence of both nalidixic acid and H2O2 at the same time showed no synergistic responses.     

Benzo(a)pyrene quinone metabolism in tracheal organ cultures.

The C^? methyl group of methionine-29 of RNAase was enriched with ¹³C. The synthesis involved the reaction of RNAase with ¹³CH₃I at pH 4. S-Methylmethionine-29 RNAase was recovered in 80% yield. This sulfonium derivative was subsequently demethylated with 0.1 M mercaptoethanol at pH 8.5, 25°C for 4 days. These conditions allowed the demethylation reaction to successfully compete with the reaction of the thiol with the four disulfide bridges in RNAase. After dialysis, concentration and chromatography, native RNAase with approx. 50% of its Met²⁹ methyl groups enriched in ¹³C was recovered as was unreversed S-Methylmethionine-29 RNAase. Both proteins showed full enzymatic activity toward cytidine 2′:3′-cyclic monophosphate. ¹³C-methyl signals from enriched RNAase and the sulfonium derivative were observed at 13.8 and 26.7 ppm from TMS respectively. Preliminary denaturation studies with the methylated protein suggest that ¹³C enrichment of methionine methyl groups in RNAase will be a useful technique for following the unfolding transition at these sites of the protein.     

Assessment of the Dermal Bioavailability of Soil-Aged Benzo(a)pyrene

Exposure to polycyclic aromatic hydrocarbons (PAHs) in soil is a major health concern because of their mutagenic and carcinogenic properties. The aim of this research was to determine the dermal bioavailability of benzo(a)pyrene (BaP) aged in either a sandy or a clay soil in order to assess the health risks and remediation goals for the chemical. In vitro flow-through diffusion cell studies were conducted utilizing dermatomed male pig skin. The amount of radioactive chemical was measured that penetrated skin into receptor fluid and which was bound to skin following soap and water decontamination. BaP bioavailability was decreased by 95 to 98% after 3 months of aging in soil relative to the pure compound. Less than 0.3% of the dose was detected in receptor fluid for all treatments. While most of the dose was bound to skin after administering the pure compound, the majority of the radioactivity was found in the soil and decontaminate after aging. The results indicate that the health risk from exposure to BaP is significantly reduced as the compound ages in soil and that less soil cleanup would be needed at sites contaminated with aged BaP.     

Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli.

A chloramphenicol resistance gene was cloned into a plasmid-borne dut gene, producing an insertion mutation that was then transferred to the chromosome by allelic exchange. The mutation could not be acquired by haploid strains through substitutive recombination, even when two flanking markers were simultaneously transduced. The insertion was easily transferred, via generalized transduction, into the chromosomal dut region of strains harboring a lambda dut + transducing phage; however, the resulting dut mutant/lambda dut + merodiploid could not then be cured of the prophage. This apparent lethality of the mutation could not be explained by effects on adjacent genes; the dfp gene retained complementing activity, and a ttk insertion mutant was viable. The dut gene product, deoxyuridine triphosphatase, is known to reduce incorporation of uracil into DNA and to be required in the de novo synthesis of thymidylate. Therefore, an attempt was made to determine whether the dut insertion would be tolerated in strains carrying the following compensatory mutations: dcd (dCTP deaminase) and cdd (deoxycytidine deaminase), which should reduce dUTP formation; ung (uracil-DNA glycosylase), which should reduce fatally excessive excision repair; deoA (thymidine phosphorylase), which should enhance the utilization of exogenous thymidine; and sulA, which should reduce the lethal side effects of SOS regulon induction. These mutations, either alone or in various combinations, did not permit the survival of a haploid dut insertion mutant, suggesting that the dut gene product might have an essential function apart from its deoxyuridine triphosphatase activity.     

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.     

Conjugation is not required for adaptive reversion of an episomal frameshift mutation in Escherichia coli.

Adaptive reversion of a lac allele on an F' episome in a strain of Escherichia coli is dependent on the RecA-BCD pathway for recombination and is enhanced by conjugal functions. However, conjugation, i.e., transfer of the episome, whether between distinct populations of cells or between newly divided siblings, does not contribute to the mutational process.     

Suppression of a frameshift mutation in the recE gene of Escherichia coli K-12 occurs by gene fusion.

The nucleotide sequences of a small gene, racC, and the adjacent N-terminal half of the wild-type recE gene are presented. A frameshift mutation, recE939, inactivating recE and preventing synthesis of the active recE enzyme, exonuclease VIII, was identified. The endpoints of five deletion mutations suppressing recE939 were sequenced. All five delete the frameshift site. Two are intra-recE deletions and fuse the N- and C-terminal portions of recE in frame. Three of the deletions remove the entire N-terminal portion of recE, fusing the C-terminal portion to N-terminal portions of racC in frame. These data indicate that about 70% of the N-terminal half of recE is not required to encode a hypothesized protein domain with exonuclease VIII activity.     

用EROD研究苯并芘和六氯苯的联合毒性作用

用7-乙氧基异叻唑酮-脱乙基酶(EROD)检测的方法,研究了苯并芘和六氯苯对日本青鳉肝脏EROD酶的比活力的影响。结果表明,苯并芘和六氯苯对EROD酶的比活力均有激活作用,在实验浓度范围内,EROD酶的比活力与两者浓度之间存在剂量-效应关系。苯并芘和六氯苯表现为一定的协同作用。实验同时发现日本青鳉在六氯苯和苯并芘中暴露后,EROD酶的比活力开始有一个短暂的降低,然后持续升高。对六氯苯和苯并芘暴露的最佳时间进行了探讨。     

The Escherichia coli tppB (ydgR) gene represents a new class of OmpR-regulated genes

The EnvZ/OmpR two-component regulatory system plays a critical role in the Escherichia coli stress response. In this study, we examined the expression of a new OmpR-regulated gene, ydgR. Our results indicate that ydgR is equivalent to the Salmonella enterica serovar Typhimurium tppB gene and represents a new class of OmpR-regulated genes.     

Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA-cleaving antibiotic bleomycin and the DNA-alkylating agent ethyl-methanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co-operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.