Toxic and phototoxic effects of tetraphenylporphinesulphonate and haematoporphyrin derivative in vitro

The toxic and phototoxic effects of tetraphenylporphinesulphonate (TPPS4) and haematoporphyrin derivative (HpD) have been examined in vitro. TPPS4 was found to have less dark toxicity to the cells than HpD as measured by inhibition of cell multiplication and colony formation at comparable extracellular concentrations. TPPS4 was also less effective than was HpD in photoinactivating NHIK 3025 cells by more than a factor 2 which should be expected on the basis of cellular uptake. Spectrofluorometric data suggest that HpD in cells interacts more with lipids than TPPS4. This might explain the large photosensitizing effect of HpD compared to TPPS4 since the lifetime of singlet oxygen is about a factor of 10 longer in a lipid environment than in an aqueous environment. The uptake of TPPS4 and HpD by cancer cells in vitro does not correlate with previous in vivo data, indicating retention of TPPS4 in the tumour stroma. This makes in vitro/in vivo extrapolation difficult with regard to the use of TPPS4 as an agent for photodynamic therapy.     

Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells

Novel N-triazolyl maleimide derivatives were synthesized by azide–alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100 µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.     

Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts.

Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.     

Cytotoxic and mutagenic effects of emodin on cultured mouse carcinoma FM3A cells

Employing a suspension culture of a mouse mammary carcinoma cell line, FM3A cells, the cytotoxicity and induced mutagenicity of emodin (EM) were examined and compared with those of 2-hydroxy-emodin (2-OH-EM), which was identified as an active form of EM in the Ames/microsomes assay. EM was cytotoxic to FM3A cells in concentrations of 1-10 micrograms/ml, and induced 6-thioguanine-resistant (6TGr) mutation. 2-OH-EM was a little more toxic than EM, but induced little mutation.     

Cytotoxic activities of normal cultured human T cells.

Normal human T cells grown in continued cultures in medium containing conditioned medium (CM) from PHA-stimulated lymphocytes were studied for their ability to manifest three known forms of cell-mediated cytotoxicity: lectin-induced cellular cytotoxicity (LICC), natural killer cell (NK) activity, and antibody-dependent cellular cytotoxicity (ADCC). The cultured T cells (CTC) were very effective mediators of LICC, being cytotoxic even at very low attacker-target cell ratios in the presence of different lectins, and against different types of targets. When tested without the addition of lectin, the CTC demonstrated a low degree of spontaneous cytotoxicity. This spontaneous cytotoxicity might not be due to conventional NK cells however, since the CTC failed to show significant numbers of cells with Fc receptors (FcR) for IgG, and had no detectable ADCC activity. CTC could represent a population enriched in polyclonal activated T cells with low spontaneous cytotoxicity against a variety of allogeneic target cells, which is greatly enhanced by the addition of lectins dur ing the 51Cr release assay.     

Separation and analysis of haematoporphyrin derivative components by high-performance liquid chromatography

High-performance liquid chromatography has been used to separate and analyse the components of haematoporphyrin derivative, a material used in cancer phototherapy. Both haematoporphyrin derivative in the solid form and the solution derived from it have been quantitatively analysed on reversed-phase columns. The factors (low pH, presence of ion-pairing reagent and solvent) that are of importance in optimising these separations are discussed.     

Cytotoxic effects of aflatoxin B1 and its association with cellular components in chicken embryo primary cultured cells.

We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.     

Photosensitivity of DNA replication and respiration to haematoporphyrin derivative (photofrin II) in mammalian CV-1 cells

DNA synthesis, as well as respiration, has been studied in CV-1 cells incubated with 5 or 25 micrograms/cm3 haematoporphyrin derivative Photofrin II (PF II) for 1, 24 or 48 h and then irradiated with various doses of UVA light (365 nm). The impairments of DNA synthesis increased with the duration of incubation with the porphyrin, its concentration and the dose of irradiation. The cellular consumption of oxygen is also inhibited by the treatment, but less severely. In the case of the higher PF II concentration (25 micrograms/cm3), the impairment of DNA synthesis after illumination seems to be mainly due to 3HTdR transport inhibition. This effect can be related to plasma membrane damage as shown by lactate dehydrogenase leakage. At 5 micrograms/cm3 PF II, DNA synthesis inhibition is observed even after short exposure to PF II and light without 3HTdR transport impairment. In that case, DNA and/or mitochondrial photodamage may explain the inhibition.     

Cytotoxic effects induced by oxidative stress in cultured mammalian cells and protection provided by Hsp27 expression

There is currently great interest in the development of methods to analyze intracellular redox state and the cellular damages generated by oxidative stress. General methods for analyzing reactive oxygen species and glutathione level are presented together with more recently developed protocols to analyze the consequences of oxidative stress on the oxidation of macromolecules. Finally, techniques to study modalities of constitutive expression of Hsp27 in mammalian cells are considered as well as methods used to determine the protective activity of this small heat shock protein against the deleterious effects induced by oxidative stress.     

Photosensitization with derivatives of haematoporphyrin

This review describes recent progress in delineation of the structure of the active component(s) in the tumour-localizing photosensitizer HPD (haematoporphyrin derivative), along with suggestions concerning the likely determinants of accumulation of this product by different tissues.     

Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured Rhesus kidney cells

Cultured Rhesus kidney cells were incubated in the 7.5 and 15% solutions at 4 and 25 ° C and for periods ranging from 10 to 60 min.The ultrastructural alterations, while varying from cell to cell, were evident even following the 10-min incubation interval. The most frequent and least severe structural changes included lipid accumulation, increased number of lysosomes, dilation and degranulation of rough surfaced endoplasmic reticulum, as well as the swelling of mitochondria and damage to mitochondrial membranes.The more severe instances of cellular lesions were represented by the extensive damage to cell membranes, karyorrhexis, total obliteration of the internal structure of mitochondria, and areas of complete loss of hyaloplasm.The extent and frequency of cellular lesions appeared to be determined primarily by the concentration of DMSO with the temperature and duration of incubation being important ancillary determinants.     

Cytotoxic and mitogenic effect of antimicrobial peptides from neutrophils on cultured cells

Cytotoxic and mitogenic activities of human and rabbit defensines (HNP and NP-2, resp.) and pig antimicrobial peptides from leukocytes (PR-39, prophenin PF-2 and protegrin PG-2) were studied. The above peptides were added to serum-free cell culture medium of the target cell lines K562, L929 and Hep22a. Cytotoxicity was estimated within 1, 3, 6, 24 and 48 h of cell incubation with the tested peptides in concentrations 1, 10, 25 or 100 micrograms/ml. All the examined peptides exhibited a distinct time- and concentration-dependent cytotoxicity. Moreover, by contrast to pig peptides, defensines could induce proliferation in cell subpopulations from cell lines L929 amd Hep22a, or L929 (defensines HNP and NP-2, resp.), keeping resistance to their cytotoxic action.     

Cytotoxic effects of NSL-1406, a new thienopyrimidine derivative, on leukocytes and osteoclasts

We synthesized a series of thienopyrimidine derivatives and examined their cytotoxic effects on several cell lines. One of the derivatives, NSL-1406, was shown to exert potent cytotoxic effects on leukemia cell line including P388 cells and J774 cells. It was also inhibitory on mouse osteoclasts and suppressed the in vitro bone resorption by osteoclasts at nanomolar concentrations.     

Prooxidant effects of beta-carotene in cultured cells

There is a growing body of interest on the role of β-carotene and other carotenoids in human chronic diseases, including cancer. While epidemiological evidence shows that people who ingest more dietary carotenoids exhibit a reduced risk for cancer, results from intervention trials indicate that supplemental β-carotene enhances lung cancer incidence and mortality among smokers. A possible mechanism which can explain the dual role of β-carotene as both a beneficial and a harmful agent in cancer as well as in other chronic diseases is its ability in modulating intracellular redox status. β-Carotene may serve as an antioxidant or as a prooxidant, depending on its intrinsic properties as well as on the redox potential of the biological environment in which it acts. This review summarizes the available evidence for a prooxidant activity of β-carotene in cultured cells, focusing on biochemical and molecular markers of oxidative stress, which have been reported to be enhanced by the carotenoid.     

Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.     

Cytotoxic effects of zirconium oxychloride on bone marrow cells of mice

A single oral administration of an aqueous solution of zirconium oxychloride to mice of both sexes in concentrations $\text{1}\text{20}$, $\text{1}\text{6}$, $\text{1}\text{2}$ of LD₅₀ induced chromosomal abnormalities in bone marrow cells. The frequencies of aberration were directly proportionate to the concentrations used. Female mice were found to be more susceptible than male mice, though not to a significantly higher level. This is the first report on the clastogenicity of this metal.     

Cytotoxic effects of thiopyrimidines

The cytotoxic action of 2-thiouracil, 2-thiocytosine, 2-thiouridine and 4-thiouridine was studied in cultures of a clone of Chinese hamster cells with a generation time of 16 hours (S — 8 hours, G₂ — 2 hours, and G₁ plus M — 6 hours). The cells were synchronized at metaphase by the method of reversal of colcemid inhibition and cell survival was measured by their colony-forming ability. The four analogs induced cytotoxic effects which increased with the concentration of the chemical and the length of the exposure time. Exposure to 4 × 10^?4 M 2-thiocytosine, 2-thiouridine or 4-thiouridine for a period of 20 hours reduced cell survival to less than 10% of the controls. The other analog (2-thiouracil) was less effective when tested at similar concentrations and time of exposure and decreased the survival to only 35% of the controls. Short periods of treatment (one hour) produced little effect at concentrations of 4 × 10^?5M, and affected the survival of cells differently when 4 × 10^?4 M were administered at different stages of the cell cycle. Two peaks of maximum sensitivity, one at late G₁ and the other at G₂ were observed. These peaks correspond to the peaks of maximum RNA synthesis described for synchronized mammalian cells. Therefore, it is likely that the cytotoxic effects of thiopyrimidine analogs are related to interference with RNA synthesis.