Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene.

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.

BCL2 gene aberration as an IPI-independent marker for poor outcome in non-germinal-centre diffuse large B cell lymphoma.

AIM: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the western hemisphere, and is characterised by a highly variable outcome that impedes individual risk assessment. Lacking reliable biomarkers, the international prognostic index (IPI) has been the most reliable factor to predict survival and stratify patients for therapy. The aim of this study was to investigate the frequency and potential prognostic role of BCL2 aberrations on the chromosomal level and the protein level in a large DLBCL collective. METHODS: Fluorescence in situ hybridisation (FISH) with commercially available dual-colour break-apart probes and immunohistochemistry were used to assess BCL2 gene abnormalities and bcl2 protein expression on validated tissue microarrays containing 224 well-characterised cases of primary DLBCL. RESULTS: FISH analysis of BCL2 revealed a break in 40/215 cases (19%) and a gain in 66/171 (39%) cases. Only BCL2 gains correlated with bcl2 protein expression (p = 0.001). Presence of any BCL2 gene abnormality, particularly gains, correlated independently of the IPI with a significantly worse prognosis in DLBCL of non-germinal centre (non-GC) phenotype as opposed to DLBCL of non-GC type without this genetic alteration (p = 0.003). DLBCL of germinal centre phenotype did not show this association. CONCLUSIONS: Cases of DLBCL of the non-GC type with BCL2 gene aberration are accompanied by a significantly worse prognosis as opposed to cases without such gene abnormalities. It may be helpful to asses BCL2 gene abnormalities by FISH in addition to assessing established parameters for individual risk estimation in DLBCL.

Aberrations of the MYC gene in unselected cases of diffuse large B-cell lymphoma are rare and unpredictable by morphological or immunohistochemical assessment.

Diffuse large B-cell lymphomas (DLBCL) with aberrations of MYC probably represent a distinct clinicopathological entity following an aggressive course. Their incidence in unselected DLBCL collectives is debatable and the identification of such cases may be difficult. Therefore, the molecular epidemiology of MYC aberrations in DLBCL and whether they can be predicted by morphology and immunohistochemistry were studied on tissue microarrays containing 333 cases. Evaluation of MYC by fluorescence in situ hybridisation was successful in 220/333 (66%) cases. 9/220 (4%) cases showed MYC breaks. Re-evaluation of these tumours did not show any specific morphological and/or immunohistochemical features. The median survival time was 9 months for the respective patients, as opposed to 80 for patients without MYC breaks. The presence of MYC breaks in DLBCL cannot be reliably predicted by conventional methods. Since such patients might profit from different forms of treatment, routine testing of all DLBCL for MYC aberrations is suggested.

Phototoxicity and photogenotoxicity of nine pyridone derivatives.

Nine structurally related pyridone derivatives were assayed for photogenotoxicity and phototoxicity in the Ames test, the chromosomal aberration test in V79 cells and the neutral red uptake (NRU) test in 3T3 cells. All nine compounds absorb light to a comparable degree at wavelengths between 380 and 430 nm. Seven of the nine compounds were found to produce high quantities of singlet oxygen (1O(2)) upon irradiation in the presence of oxygen. These seven compounds were highly phototoxic in the NRU test, three were clearly and two were marginally photomutagenic in the Ames test, five were assessed as clearly and two as equivocally photoclastogenic in the chromosomal aberration test. Two compounds showed substantially lower 1O(2) yields. The pyridone ring of these two compounds is attached to a non-aromatic ring, while for the seven other compounds the chromophore system including the pyridone ring consists of two or three aromatic rings. One of the two compounds with low 1O(2) yields was distinctly less phototoxic and did not induce photogenotoxic effects. The other, structurally an indolo derivative and not the common thieno derivative, was, however, similarly phototoxic as the seven compounds with high 1O(2) quantum yield and was also clearly photogenotoxic indicating that different action pathways, not involving singlet oxygen, have to be considered at least for this compound.

The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992-93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994-95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for "In Vitro Phototoxicity Testing", incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.

Antimalarial activity of the bisquinoline trans-N1,N2-bis (7-chloroquinolin-4-yl)cyclohexane-1,2-diamine: comparison of two stereoisomers and detailed evaluation of the S,S enantiomer, Ro 47-7737.

The S,S enantiomer of the bisquinoline trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine, Ro 47-7737, is significantly more potent against chloroquine-resistant Plasmodium falciparum than the R,R enantiomer and the previously described racemate. Both the enantiomers and the racemate are more potent inhibitors of heme polymerization than chloroquine, and their activities are probably mediated by inhibition of this parasite-specific process. The S,S enantiomer, Ro 47-7737, was studied in more detail and proved to be a potent antimalarial in the treatment of P. vivax ex vivo and P. berghei in vivo. Its suppression of P. berghei growth in a mouse model (50% effective dose, 2.3 mg/kg of body weight) was equal to that of chloroquine and mefloquine, and Ro 47-7737 was found to be more potent than these two drugs in the Rane test, in which the curative effect of a single dose is monitored. The dose at which 50% of animals were permanently cured (34 mg/kg) was markedly superior to those of chloroquine (285 mg/kg) and mefloquine (> 250 mg/kg). When administered orally at 50 mg/kg, Ro 47-7737 also showed a faster clearance of parasites than either chloroquine or mefloquine, and unlike the other two compounds, Ro 47-7737 showed no recrudescence. In a study to compare prophylactic efficacies of oral doses of 50 mg/kg, Ro 47-7737 provided protection for 14 days compared to 3 days for mefloquine and 1 day for chloroquine. The good curative and prophylactic properties of the compound can be explained in part by its long terminal half-life. The ability to generate parasite resistance to Ro 47-7737 was also assessed. With a rodent model, resistance could be generated over eight passages. This rate of resistance generation is comparable to that of mefloquine, which has proved to be an effective antimalarial for many years. Toxicity liabilities, however, ruled out this compound as a candidate for drug development.

Low ICAM-1 expression in the epidermis of depigmenting C57BL/6J-mivit/mivit mice: a possible cause of muted contact sensitization.

The depigmenting C57BL/6J-mivit/mivit) (mivit/mivit) mouse, a congenic mutant of the C57BL/6 strain, exhibits an isolated, single immune deficiency. It is unable to mount a normal immune/inflammatory response upon epicutaneous application of DNFB or TNCB, although it does respond normally to oxazalone. The present investigations have been carried out to further study this deficiency. In vivo, C57BL/6 mice could be sensitized by the epicutaneous application of the hapten TNCB, the subcutaneous injection of hapten(TNBS)-conjugated C57BL/6, and hapten conjugated mivit/mivit epidermal cells. In the mivit/mivit mice, however, only subcutaneous injection of haptenized C57BL/6 epidermal cells caused an immune response. The response of these mivit/mivit mice could be documented only by adoptive transfer of splenic lymphocytes into naive C57BL/6 animals which then reacted to challenge doses of TNCB. These observations suggest that mivit/mivit epidermal cells can process and present and mivit/mivit T lymphocytes can react to the antigen. We postulated the presence of a deficient in vivo interaction between epidermal cells and T lymphocytes in the mivit/mivit mice. ICAM-1 is an important adhesion signal regulating epidermal cell/T-lymphocyte interaction. Its expression in mivit/mivit mice was studied using YN1/1 antibody against MALA-2, the murine counterpart of human ICAM-1. In contrast to C57BL/6 animals, the mivit/mivit epidermis essentially did not stain with the antibody after hapten challenge. In vitro after stimulation with TPA or IFN-gamma, the mivit/mivit epidermal cells expressed significantly lesser amounts of ICAM-1 than the C57BL/6 epidermal cells. Lower expression of ICAM-1 by mivit/mivit epidermal cells has also been demonstrated both by direct staining and by flow cytometry. The binding of lymphocytes to mivit/mivit epidermal monolayers, which were stimulated to express ICAM-1 by IFN-gamma, was decreased compared to that of C57BL/6 epidermal cells. We conclude that the muted contact sensitization response detected in vivo in the mivit/mivit mice at least partly results from lower expression of ICAM-1 and thus defective epidermal cell/T-lymphocyte interaction.

Effect of BN 52021, a platelet activating factor antagonist, on dithranol-induced inflammation.

The effect of BN 52021, a selective platelet activating factor (PAF) antagonist was studied on dithranol-induced irritant dermatitis. Pretreatment of the skin with 0.45% BN 52021 ointment significantly suppressed the decrease in capillary resistance, rise in skin temperature and increase in skin-fold thickness produced by dithranol.

Effect of tiacrilast, a mast cell mediator-release inhibitor, on murine experimental contact dermatitis.

The effect of tiacrilast, a mast cell mediator-release inhibitor, was studied in dinitrofluorobenzene-induced allergic and croton oil- or dimethyl sulfoxide-induced irritant murine contact dermatitis. At 1% concentration, the compound significantly reduced the ear swelling in both allergic and irritant dermatitis and preserved the mast cell architecture on histopathology. These findings suggest that mast cells participate in the elicitation of murine contact dermatitis.

Comparative studies of human eosinophil migration towards platelet-activating factor and leukotriene B4.

Peripheral-blood polymorphonuclear cells from 36 donors with or without eosinophilia were studied for their in vitro responsiveness towards a wide range of concentrations of leukotriene B4 (LTB4) and platelet-activating factor (PAF). The mean percentage of migrated eosinophils was 17.6 for PAF, 21.1 for LTB4, 20.1 for buffer controls with cells from eosinophil patients, and 1.1 for PAF, 8.9 for LTB4 and 3.2 for buffer control with noneosinophil donors. The quantitative response of eosinophils towards PAF was lower than that towards LTB4 on a molar basis. The data showed high interindividual variations for eosinophil responsiveness towards mediators and buffer and suggest disease-dependent alterations of receptor expression or of basic metabolic activity of eosinophils as possible reasons for this variability.

Pharmacological studies on dithranol-induced irritative dermatitis in mice.

The effects of different pharmacological substances on dithranol-induced irritative dermatitis were studied in mice. Pretreatment of the animals with a specific platelet activating factor (PAF) antagonist, BN 52021, significantly reduced the ear swelling in a dose-dependent manner. The cyclooxygenase inhibitor indomethacin, the antihistamine clemastine, and the anti-oxidant superoxide dismutase also proved to be effective in the reduction of the dermatitis. The results provide evidence of the coinvolvement of PAF, prostaglandins, histamine, and reactive oxygen radicals in dithranol-induced irritative dermatitis in mice.

[The irritant effect of dithranol using capillary resistance measurement]. / Untersuchung der irritativen Wirkung des Dithranols mittels Kapillarresistenzmessung.

The effect of topically applied dithranol on the capillary resistance (CR) was studied both in healthy volunteers and in clinically unaffected skin of psoriatic patients. The CR of the psoriatic patients was significantly lower than that of the healthy persons. O.10% and 0.25% dithranol ointment induced a significant decrease of CR in the skin of both groups. This decrease was prevented or at least reduced, if the patients had previously been treated with either the antihistamine clemastine or the cyclooxygenase-inhibitor indomethacin. The effect of dithranol on the CR was also moderated, if 5% coal tar had been added to the dithranol preparation.

Effect of BN 52021, a platelet activating factor antagonist, on experimental murine contact dermatitis.

A specific platelet activating factor (PAF) receptor antagonist, BN 52021, was tested for its efficacy in modulating DNFB-induced allergic and croton oil-induced irritant contact dermatitis in mouse ears. Oral treatment of the animals in the elicitation phase of the dermatitis caused a significant suppression of DNFB-induced ear swelling. The compound was less active in croton oil-induced irritant dermatitis and ineffective when given during the sensitization phase of allergic dermatitis. The results provide indirect evidence for the involvement of PAF in the effector phase of murine contact dermatitis of the allergic type and also the irritant type to a lesser extent.

Involvement of arachidonate-dependent lipid mediators and platelet-activating factor in experimental murine contact dermatitis.

A 5-lipoxygenase inhibitor (5-LPI) and a platelet-activating factor antagonist (PAF-A) were studied in dinitrofluorobenzene (DNFB)-induced allergic and croton-oil-induced irritant murine contact dermatitis. Both inhibitors, at 1 and 5% concentrations, significantly reduced the ear swelling in allergic dermatitis while irritant dermatitis was far less affected. This suggests that 5-lipoxygenase-dependent mediators and PAF are involved in allergic contact dermatitis and that these mediators play only a minor role in irritant dermatitis.

Studies on the role of leukotrienes in murine allergic and irritant contact dermatitis.

A specific peptidoleukotriene receptor antagonist, Ro 23-3544, was tested for its efficacy in modulating DNFB-induced allergic and croton oil-induced irritant contact dermatitis in mouse ears. Treatment shortly after elicitation of the dermatitis, and for up to 5 days thereafter, was moderately effective in suppressing DNFB-induced ear swelling in a dose-dependent fashion. Daily pre-treatment of the ears for 1 week caused a more marked reduction of DNFB-induced ear swelling during the first 48 h after elicitation. No reduction, but rather an increase in ear swelling was observed with croton oil-induced dermatitis. These results indicate that peptidoleukotrienes play a role in the early stages of elicitation of murine allergic, but not irritant contact dermatitis and that a specific receptor antagonist can only partially reverse the effect of peptidoleukotrienes once the dermatitis is established.