ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

PHOTOREACTIVATION IN A phrB MUTANT OF Escherichia coli K-12: EVIDENCE FOR THE ROLE OF A SECOND PROTEIN IN PHOTOREPAIR

Abstract In Escherichia coli , the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phr B, another locus phr A having been proposed in association with PR. The relevance of the putative phr A gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosphila melanogaster . which specifically repairs pyrimidine (6–4) pyrimidone photoproducts ([6–4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6–4) photoproduct, has cast new light on the prospective role of phr A in PR. We have determined the nucleotide sequence of the putative phr A gene, which suggests it codes for a protein of 38 kDa. When the putative phr A gene was cloned into an expression vector and transformed into a phr A phr B mutant of E. coli , a level of photorepair was observed, which could correspond to repair of (6–4) photoproducts.     

PHOTOCHEMICAL REACTIONS OF CHLORPROMAZINE; CHEMICAL AND BIOCHEMICAL IMPLICATIONS

Abstract— The photoexcited chlorpromazine reacts with methanol to yield promazine and 2-methoxypromazine by two different reaction pathways: hydrogen atom abstraction and nucleophilic attack. respectively. When the photoexcitation of chlorpromazine is performed in the presence of protein or nucleic acids, chlorpromazine binds to the biopolymer. This binding is drastically pH-dependent and correlates to the phototoxic effect exhibited in chlorpromazine—photosensitization of E. coli. No photodynamic damage of E. coli attributed to CPZ-sensitization of molecular oxygen could be detected.     

THE EXPRESSION OF ENTEROTOXIN A~-B~+ GENE OF V. cholerae IN E. coli

The cloning in E. coli of a cholerae toxin gene that is A~-B~+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.     

THE ROLE OF SOLAR ULTRAVIOLET RADIATION IN 'NATURAL' WATER PURIFICATION

Abstract— The concentration of Escherichia coli in the input and output of a tertiary wastewater system (4 lagoons) has been monitored over an 11 month period. The integrated flux of biologically active solar ultraviolet (UV) radiation was measured during this period. By also determining (1) the effective temperature in the system, (2) the growth rate of E. coli at the effective temperature, (3) the penetration of the solar UV into the lagoons, (4) the dose-response relation for killing of E. coli by UV and (5) the retention time of water in the system, it is possible to compare the 'die off' expected from solar UV exposure to the actual 'die off' observed for different batches of water.
The observed killing of E. coli was quite close to the values calculated, considering the numerous factors involved. Solar UV light would thus seem to be a very important factor in the natural purification of water. Because each successful species must possess characteristics (physiological or behavioral) which provide adequate resistance to solar UV, the ecological role of solar UV radiation has not been widely appreciated.     

PHOTOBINDING OF CHLORPROMAZINE AND ITS SULFOXIDE IN VITRO AND IN VIVO

Abstract— Photosensitivity as observed after chlorpromazine (CPZ) treatment is enhanced in the UVA- rather than the UVB region, whereas CPZ has its absorption maximum at 305 nm. This long wavelength sensitivity has sometimes been ascribed to CPZ-sulfoxide (CPZSO) which has an absorption maximum at 340 nm. We compared the photobinding properties of CPZSO and CPZ under both in vitro and in vivo conditions.
With 310 and 370 nm lamps CPZSO absorbs twice as much light as CPZ but still binds less efficiently to HSA in vitro. At wavelengths longer than 380 nm CPZSO does not absorb nor photobind to HSA (420 nm lamps) in contrast to CPZ.
In vivo the bioavailability of CPZ and CPZSO in ears, eyes and skin of the back of Wistar rats is comparable, yet irradiation with 370 nm light caused more CPZ-photobinding in these tissues. Chlorpromazine binds relatively more compared to CPZSO, to constituents of deeper lying tissues (dermis). This corresponds with the observation that both the ratio of in vitro CPZ photobinding to CPZSO photobinding, and the penetrating ability of light in the skin increase with wavelength.
In the eyes, where the lens efficiently filters out light with wavelengths shorter than 370 nm, no CPZSO photobinding was observed, in contrast to CPZ; this also corresponds with the in vitro experiments. Therefore it seems more likely that the observed wavelength maximum in the photosensitivity action spectrum after CPZ treatment should be attributed to the non-sulfoxidated drug rather than to the sulfoxidated compound.     

ROLES OF THE RELA+ GENE AND OF 4-THIOURIDINE IN NEAR-ULTRAVIOLET (334 nm) RADIATION INHIBITION OF INDUCED SYNTHESIS OF TRYPTOPHANASE IN ESCHERICHIA COLI B/r

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) is known to inhibit the induced synthesis of tryptophanase by tryptophan in Escherichia coli , showing an action spectrum similar to that for near-UV-induced growth delay. The present work shows that a rel A mutant of E. coli B/r exhibits 50% as much monochromatic near-UV (334 nm) inhibition of tryptophanase induction as the wild type, and that a mutant lacking 4-thiouridine, an unusual nucleoside in tRNA, exhibits < 10% as much inhibition of tryptophanase induction. These findings indicate that 4-thiouridine is almost the sole chromophore for this effect in E. coli B/r, but that only 50% of the effect operates by a mechanism utilizing the rel A ⁺ gene product; growth delay appears not to be primarily involved.     

In vivo PHOTODEGRADATION OF CHLORPROMAZINE

The in vivo photodegradation of chlorpromazine (CPZ) in the skin was investigated after systemic administration of 3H-CPZ to shaven Wistar rats and exposure to UV-A. Promazine (PZ) and 2-hydroxy-promazine (2-OH-PZ) appeared to be formed in irradiated rats, but not in the skin of rats kept in the dark. This indicates that upon irradiation with UV-A the PZ-radical is formed which can be held responsible for the photobinding to eye and skin constituents as observed earlier [Schoonderwoerd and Beijersbergen von Henegouwen (1987) Photochem. Photobiol. 46, 501-505]. Chlorpromazine-sulfoxide (CPZSO) is a major metabolite of CPZ. Less CPZSO was found in the skin of irradiated rats compared to those kept in the dark. As this appeared not to be caused by photobinding or photodegradation of CPZSO it can be concluded that CPZSO is not a photoproduct of CPZ under these experimental conditions. This study shows that the in vivo photodegradation of CPZ proceeds via the promazinyl radical rather than via the radical cation.     

In vitro PHOTODEGRADATION OF CHLORPROMAZINE

The in vitro photodecomposition of chlorpromazine (CPZ) was investigated with the aim to evaluate possible reactive determinants that could play a role in the occurrence of the in vivo -observed photosensitivity. In view of the in vivo situation, CPZ was dissolved in low concentration in buffered aqueous solution (pH 7.4) or in dilute human serum and irradiated with low intensity (5–7 W m^-2) UV-A and UV-B. No distinct difference was found between UV-A or UV-B irradiation as far as photoproduct formation is concerned. This suggests the same degradation mechanism at both wavelength ranges. In buffered aqueous solution, irradiation of CPZ resulted in 65 and 90% 2-hydroxypromazine (PZOH), 5 and 7% promazine (PZH) and 2 and 0% chlorpromazinesulfoxide (CPZSO) under aerobic and anaerobic conditions, respectively. In dilute human serum, there was only a shift in the PZH/PZOH ratio, probably as a result of H-atom or electron donation by sulfur containing groups present in proteins. The results demonstrate that photodegradation of CPZ in vitro , under conditions relevant to the in vivo situation, proceeds almost entirely by dechlorination rather than by radical cation formation (the essential pathway of CPZSO production). Thus we conclude that the thiyl radical cation probably does not play a major role in the in vivo -observed phototoxic reactions.     

THE ROLE OF DNA POLYMERASE I IN LIQUID HOLDING RECOVERY OF UV-IRRADIATED ESCHERICHIA COLI

Abstract. –Excision of cyclobutyl dipyrimidines from, and accumulation of strand interruptions in, DNA of different strains of E. coli K12 were determined during liquid holding recovery after UV irradiation. The extent of Pyr <> Pyr excision was the same (20–25%) for both a pol A mutant ( E. coli P3478) and its parental wild type strain ( E. coli W3110); however, single strand interruptions accumulate during liquid holding of polA cells, but not in the parental strain. In contrast, excision was greatly reduced in a mutant (KMBL 1789) which is defective in the 5'→3' exonucleolytic function of DNA polymerase I. These data suggest that excision and resynthesis during liquid holding are carried out primarily, if not entirely, by DNA polymerase I. We further conclude that excision alone is both a necessary and sufficient condition to elicit liquid holding recovery, and that this excision requires a functional polymerase I 5'→ 3' exonuclease.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

COMPARISON OF SURVIVAL OF A U.V.-IRRADIATED URACIL-REQUIRING STRAIN OF ESCHERICHIA COLI B IN THE PRESENCE OF CHLORAMPHENICOL AND IN THE ABSENCE OF THE ESSENTIAL METABOLITE

Abstract— Restoration of viability of E. coli B U- after a total dose of 900 ergs/mm2 U.V. in the presence of chloramphenicol and in the absence of uracil was studied. Both treatments result in the recovery of the same fraction of the irradiated bacterial population. There is no additive effect if both treatments are applied to the same irradiated culture.     

CHARACTERIZATION OF RAD4 GENE REQUIRED FOR ULTRAVIOLET-INDUCED EXCISION REPAIR OF Saccharomyces cerevisiae PROPAGATED IN Escherichia coli WITHOUT INACTIVATION

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J. Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

INVESTIGATIONS ON THE MECHANISM OF CHLORPROMAZINE PHOTOTOXICITY: EFFECTS ON LYSOSOMES OF CULTURED HUMAN FIBROBLASTS

Abstract The effect of chlorpromazine (CPZ) and UVA on lysosomes of cultured normal human fibroblasts has been investigated. Acid phosphatase (ACPase) activity in 12 000 g pellet of cells treated with CPZ (10 μg/m l ) and UVA (6 × 10⁴ J/m²) was found to be decreased as compared with non-treated, CPZ or UVA treated control cells. This decrease, however, was not accompanied by a concomitant increase in ACPase activity in the 12 000 g supernatant. The addition of Triton X-100 to cells pretreated with CPZ + UVA resulted in only a moderate increase in ACPase activity of the 12 000 g supernatant. ACPase activity of the cells incubated in media containing preirradiated CPZ was also found to he decreased. These results indicate that CPZ + UVA directly inactivate lysosomal enzymes, possibly without affecting the membrane.     

EXPRESSION OF AN Anacystis nidulans PHOTOLYASE GENE IN Escherichia coli; FUNCTIONAL COMPLEMENTATION AND MODIFIED ACTION SPECTRUM OF PHOTOREACTIVATION

The Anacystis nidulans photolyase gene inserted in an expression vector plasmid was introduced into Escherichia coli cells and the production of Anacystis photolyase protein was confirmed by reaction with antibodies raised against photolyase purified from A. nidulans cells. The Anacystis photolyase functioned in photoreactivation repair defective E. coli cells. The E. coli transformants exhibited an action spectrum with a maximum around 380 nm similar to that of E. coli photolyase in contrast with the action spectrum of A. nidulans cells which has a maximum at 437 nm. These results indicate that the Anacystis photolyase produced in E. coli cells has enzymatic activity in spite of the apparent lack of its intrinsic 8-hydroxy-5-deazaflavin cofactor.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

ROLE OF DYE MOLECULES REMAINING OUTSIDE THE CELL DURING PHOTODYNAMIC INACTIVATION OF ESCHERICHIA COLI IN THE PRESENCE OF ACRIFLAVINE

Abstract— Photodynamic inactivation of cells is caused by damage to the regions proximal to the cell envelope or to the DNA via a singlet oxygen mechanism. For penetrating dyes the possibility of either type of damage remains. The contribution of a penetrating dye. acriflavine. remaining outside E. coli B/r cells during irradiation. towards photodynamic inactivation was investigated. It was found that this contribution was either nil or negligible.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.