Chromosome-damaging effects of heraclenin in human lymphocytes in vitro

Heraclenin, a furocoumarin with an epoxide group in its side chain, was analyzed to see if it induced structural chromosome aberrations and sister-chromatid exchanges (SCEs) in human lymphocytes in vitro. The results were compared directly with those of imperatorin, which differs from heraclenin only in lacking an epoxide group. An equally strong clastogenic effect was found for both heraclenin and imperatorin: the number of metaphases with breaks was increased in both cases by approximately a factor of 6. Heraclenin produced a considerable dose-dependent increase in the SCE rate, i.e., by about 60 induced SCEs/metaphase, whereas imperatorin induced only about 4 SCEs/metaphase. The results are discussed with respect to the occurrence of structural aberrations, which are primarily due to the basic furocoumarin structure itself, whereas the large increase in the SCE rate produced by heraclenin is most probably significantly influenced by its epoxide group.     

Linear furocoumarins and other constituents from thamnosma texana

Eight linear furocoumarins and three coumarins were isolated and identified from Thamnosma texana. They were xanthotoxin, imperatorin, bergapten, alloimperatorin methyl ether epoxide, heraclenin, isopimpinellin, psoralen, oxypeucedanin, and the coumarins herniarin, osthol and thamnosmin. The linear furocoumarins appear to be agents that account for the known photosensitizing properties of Thamnosma texana, and consequently its colloquial name, ‘blisterweed.’ This is the first report on the occurrence of imperatorin, heraclenin, oxypeucedanin, herniarin or osthol in any Thamnosma species.     

Mutagenic compounds in an extract from Rutae Herba (Ruta graveolens L.). II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba

A commercial tincture prepared from Rutae Herba (Ruta graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of Chlamydomonas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen, imperatorin, and 3 furoquinoline alkaloids (dictamnine, gamma-fagarine, skimmianine) were detected. All compounds revealed photomutagenic properties but their activities were quite different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of these compounds in the tincture and their activities we conclude that bergapten is mainly responsible for the photomutagenicity of the tincture. The lower phototoxicity and photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also form biadducts.     

UV-A induces persistent genomic instability in human keratinocytes through an oxidative stress mechanism

Ultraviolet-A (UV-A, 320 to 400 nm) radiation comprises 95% of the solar ultraviolet radiation (UVR) reaching the earth's surface. It has been associated experimentally and epidemiologically with malignant melanoma. In this study we investigated whether UV-A radiation can induce a persistent, heritable hypermutability in mammalian cells similar to that observed following ionising radiation (IR). Using the immortalized human skin keratinocyte cell line HaCaT we found that UV-A radiation does lead to a continuing reduction in plating efficiency, an increased "spontaneous" mutant fraction, and an increase in micronucleus formation up to 21 d after initial exposure. Reversal of these effects using catalase may indicate a role for hydrogen peroxide in this phenomenon. These results add to the significance of UV-A radiation as a risk factor in skin carcinogenesis.     

A comparison of cytotoxicity, ouabain-resistant mutation, sister-chromatid exchanges, and nascent DNA synthesis in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Chinese hamster V79 cells were treated with either (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide I) or (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide II) and the nascent DNA was labeled with [Me-3H]thymidine. The cells were harvested for determination of cytotoxicity, sister-chromatid exchanges (SCE), ouabain-resistant (Or) mutations and the size of newly synthesized daughter-strand DNA. Both isomers caused dose-dependent decreases in survival of cells and in the size of nascent DNA. Increases in the frequencies of SCE and of Or mutation were found in cells treated with either isomer. However, B[a]P-diol epoxide I caused 10--20-fold more Or mutations and 50-100% more SCE than did B[a]P-diol epoxide II at equal molar dose levels. In contrast to the marked difference in the frequencies of both SCE and Or mutations caused by both compounds, the isomers induced similar reductions in the size of the nascent DNA at equal dose levels. In comparing the molecular and biological effects of the two isomers the reduction in the size of nascent DNA was more closely related to cytotoxicity than to the induction of SCE or Or mutations.     

Mutagenic compounds in an extract from Rutae Herba (Ruta graveolens L.) II. UV-A mediated mutagenicity in the green alga Chlamydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba

A commercial tincture prepared from Rutae Herba (Ruta graveolens L.) exhibited a moderate photomutagenicity in an arginine-requiring mutant strain of Chlamydomounas reinhardtii. In the tincture some furocoumarins, e.g., bergapten, psoralen, imperatorin, and 3 furoquinoline alkaloids (dictamnine, γ-fagarine, skimmianine) were detected. All compounds revealed photomutagenic properties but their activities were quite different. Bergapten was the most potent furocoumarin. Dictamnine, the furoquinoline with the strongest effect, reached only about 10% of the activity of bergapten. Based on the amount of these compounds in the tincture and their activities we conclude that bergapten is mainly responsible for the photomutagenicity of the tincture.The lower phototoxicity and photomutagenicity of the furoquinoline alkaloids may be due to the fact that furoquinolines form only monoadducts with DNA in the presence of UV-A in contrast to furocoumarins which also form biadducts.     

Diverse effects of three furocoumarins on human lymphocyte proliferation

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed "in vitro" by measuring 3H-thymidine (3H-TdR) incorporation in the presence and in the absence of 15-30 microM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15-30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while, unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.     

The rpoS Gene in Pseudomonas syringae Is Important in Surviving Exposure to the Near-UV in Sunlight

The near-UV component of sunlight decreased culturability of the leaf epiphyte and plant pathogen Pseudomonas syringae. Exposure of the wild-type cells for 4 h to UV-A and UV-B in sunlight was ten fold more detrimental than exposure to sunlight with just UV-A. Sensitivity to UV-A especially increased in a mutant of P. syringae lacking the global regulatory sigma factor, RpoS. No RpoS-mutant cells were culturable after 4 h of exposure to near-UV sunlight. These findings suggest that both UV-A and UV-B wavelengths cause damage to the bacterial cell and that the RpoS protein regulates protective measures for the leaf-associated pseudomonad. Received: 6 February 2001 / Accepted: 3 April 2001     

Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells

The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV-1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation.     

Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro

Background

Extracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure.

Methods

Human T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy.

Results

Cell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A.

Conclusion

8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.     

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultra-violet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m²-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01–1.0 μg/ml) for 3 different UV-A doses (3000–15000 J/m²). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks. These results suggest that the cytotoxic effects of photoactivated 8-MOP in human lymphoblastoid cells may depend on DNA interstrand crosslinks.     

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.     

Photobiophysics of furanocoumarins

Furocoumarins (psoralens) are photosensitizers of plant origin, which increase the sensitivity of biological objects to near ultraviolet (UV-A, 320-400 nm). In combination with UV-A, they are successfully used for treating many dermal and autoimmune diseases (PUVA therapy and photophoresis). Along with therapeutic effects, the furocoumarin photochemotherapy induces a number of side-effects (erythema, edema, hyperpigmentation, and premature aging of skin). All photobiological effects of furocoumarins result from their photochemical reactions. Therefore, in order to advance the therapy, it is necessary to know the photochemical mechanisms of induction of both side- and therapeutic effects. The types of photoreactions of furocoumarins classified with respect to reactive photoproducts interacting with substrate were considered. Primary emphasis was placed on reactions proceeding with the participation of photooxidation products of furocoumarins. Among these photoproducts, at least two types can be distinguished. Some of them possess membranotoxic properties, others produce the immunosuppressory action in vivo. The photochemical mechanisms of the formation of the photoproducts of furocoumarins are different. It was found that, by varying the illumination conditions (intensity of UV-A radiation or the concentration of the photosensitizer), it is possible to obtain the photoproducts of furocoumarins that have either membranotoxic or immunosuppressory properties. It was found that the mechanisms of the immunosuppressive action of the photooxidation products of furocoumarins have some features in common with those underlying the PUVA therapy and photophoresis. It is assumed that the photochemical basis of the therapeutic action of furocoumarins is the reactions with the involvement of the products of their photooxidation.     

Hypoxia and reoxygenation: A pressure for mutant p53 cell selection and tumour progression

Recent findings indicate that in a hypoxic environment, oncogenically transformed cells with a mutant form of the tumour suppressor gene p53 may have a survival advantage over similar cells with wild-type p53. This is because the extent of hypoxia-induced apoptosis has been observed to diminish with the loss of wild-type p53 function in certain cell lines. Hypoxic conditions, common in most solid tumours, may thus provide a physiological pressure to select for cells with mutations in the p53 gene. A new model incorporating cell-specific parameters is proposed here to quantify the survival advantage of mutant or null p53 cells over their wild-type counterparts at any level of oxygen deprivation. Predictions are in good agreement with previous monolayer culture experiments comparing hypoxic survival of null and wild-type p53 cells. By extending the model we are able to investigate the effects of repeated rounds of hypoxia and reoxygenation on a mixture of wild-type and mutant or null p53 cells and determine how many rounds are required before a subpopulation of mutant or null p53 cells overtakes a given population of wild-type p53 cells.     

The regulation of cytosolic epoxide hydrolase in mice

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Micronuclei and gene mutations in transgenic big Blue((R)) mouse and rat fibroblasts after exposure to the epoxide metabolites of 1, 3-butadiene

1,3-Butadiene (BD) is a commodity compound and by-product in the manufacture of synthetic rubber that elicits a differential carcinogenic response in rodents after chronic exposure. Mice are up to approximately 1000-fold more sensitive to the tumorigenicity of inhaled BD than rats, thereby confounding human risk assessment analyses. Rodent transgenic in vivo and in vitro models have been recently utilized for generating genetic toxicology data in support of risk assessment studies. However, studies have not been extended to investigate multiple endpoints of genetic damage using in vitro transgenic models. The goal of this study was to evaluate possible differences in the production of genetic damage in transgenic Big Blue((R)) mouse (BBM1) and rat (BBR1) fibroblasts exposed to three predominant epoxide metabolites of BD. Analyses of cytotoxicity, micronucleus (MN) formation, cII mutant frequency (MF) and apoptosis were assessed after in vitro exposure of BBM1 and BBR1 cells exposed to various concentrations of butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). Both BMO and DEB reduced cell survival in BBM1 and BBR1 cells. However, BDE decreased cell survival only in BBM1 cells at the concentrations evaluated. Concentration-dependent increases in the formation of MN was observed in both BBM1 and BBR1 cells, with DEB being the most potent followed by BDE and then BMO. The dose-response for mutations induced at the cII locus was essentially equal after DEB exposure of BBM1 and BBR1 fibroblasts. In contrast, the cII MF was significantly increased only in BBM1 cells after exposure to either BMO or BDE. These data demonstrate a differential genetic response for gene mutations but not for MN formation in transgenic BBM1 and BBR1 fibroblasts and suggest a rodent species-specific difference in the persistence of DNA damage that results in gene mutations. In addition, apoptosis was observed in BBR1 cells but not in BBM1 cells when treated with any of the three BD epoxide metabolites. This response may partially explain the differential response to mutations induced by BMO and BDE. These data offer insight into specific differences in mouse and rat cells with respect to their response to BD epoxide metabolites. Such data may help to explain the different tumorigenicity results observed in rodent BD carcinogenicity studies.     

Deficient repair of chemical adducts in α DNA of monkey cells

We have examined excision repair of DNA damage in the highly repeated α DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in α DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for α DNA and bulk DNA. In cells treated with furocoumarins and long-wavelength ultraviolet light, however, repair synthesis in α DNA was only 30% of that in bulk DNA, although it followed the same time course. We found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as we found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from α DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in α DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences.