Comparison of methods used for evaluation of mutagenicity/genotoxicity of model chemicals - parabens.

Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells / tissues of human origin.

The in vitro cytotoxicity of metal-complexes of porphyrin sensitizer intended for photodynamic therapy.

The sulphonated derivatives of porphyrins (e.g. TPPS4) are hydrophilic photosensitizers and have certain advantages like fully known structures and the possibility of synthetic production. The aim of this work was to study in vitro cytotoxicity and to compare the new photosensitizer MgTPPS4 with TPPS4 and its other metal-complexes (ZnTPPS4, PdTPPS4) on human skin melanom and mouse fibroblast cell lines. A photodynamic treatment was induced by light emitting diodes with three different total doses (1, 5 and 10J/cm(2)). For proper analysis and understanding of cell behavior after the administration of sensitizers, a complex battery of in vitro tests including the production of reactive oxygen species, the MTT viability test, a comet assay, a cell cycle and a type of cell death determination were used. We discovered that the most suitable photosensitizer is ZnTPPS4 because it had the biggest lethal influence on melanoma cells and the lowest lethal influence on fibroblast cells. The second most effective photosensitizer seemed to be MgTPPS4. On the basis of our results we can also assume that there is a higher accumulation of photosensitizer in a tumorous cell line. The higher concentration of photosensitizer and light dose resulted in more reactive oxygen species production and found more cells undergoing necrosis.

[Phototoxic reaction after the systemic administration of antimicrobial drugs]. / Fototoxické reakce po systémovém podaivaini antimikrobiálních léku.

Several systemically administered drugs with antimicrobial effect have undesirable phototoxic effects. This group includes some antibiotics (e.g., tetracycline, fluorochinolons, and also macrolids), sulphonamides and related chemotherapeutics, antimycotics, tuberculostatics, antiviral drugs and antimetabolites. Doctors should know the phototoxic effect of those drugs in advance, because of the possible time shortage they might have when they prescribe the drugs. The first condition is to obtain the relevant personal history data, and in case the phototoxic reaction develops, to terminate the unsuitable drug, to change it for another, to administer antihistaminics, sometimes corticoids, prevent exposition to sun, and administer local anti-inflammatory dermatological drugs.

[Use of external photoprotective agents in children living in the area of Prague]. / Pouzívání zevních fotoprotektivních prostredku u detí prazské aglomerace.

BACKGROUND: Development of skin melanoma is related to the higher exposition to ultraviolet light, namely to the repeated sun burning of children. Because no valid information on the use of photoprotection in children can be found, an extensive search on the use of such measures, their galenic form and protective features was undertaken in order to evaluate the situation and to suggest necessary improvements. METHODS AND RESULTS: A group of 140 children aged 3 to 15 years, part of the group living in Prague, another part living outside the city was examined whether they use some local photoprotection during longer exposition to sun. If the answer was positive, it was followed in which season the photoprotection is used and whether it was used repeatedly. We also questioned on the forms of the local photoprotectives and the value of solar protective factor. CONCLUSIONS: Beside the description of sun tanning characteristics of children, we were informed by their parents that 59% of children use the photoprotection regularly, 41% only occasionally. The age of the first use of protection was 3.75 years. Photoprotection was used once a day in 28% of children, repeated use (during bathing, seashore stay, tourism) was reported in 72% of cases. The galenic form of photoprotective was most frequently a sun tanning creme (42%), a sun tanning milk (30%), both forms alternatively (8%), an oil (8%) or some other form (14%). The value of photoprotective factor was reported by 108 out of 131 parents and it was less than 10 in 9 children, 11 to 22 in 60 children, 21 to 40 in 36 children and over 40 in 3 children.

[Is tamoxifene porphyrinogenic?]. / Je tamoxifen porfyrinogenní?

BACKGROUND: As reported in previous studies, porphyria cutanea tarda (PCT) developed in several tamoxifene-treated patients with breast cancer. We studied the group of patients with cancer having only tamoxifene therapy after the initial surgery. We evaluated their clinical and laboratory results and compared them with the results of the group of patients suffering also from breast tumor, but treated after the surgery with other systemic therapies, mostly with chemotherapy. METHODS AND RESULTS: 20 patients were complexly studied, 10 of them with only tamoxifene therapy, and 10 without it. Diagnosis of the breast tumor was histologically confirmed in all of them. With the use of laboratory methods we examined their urinary excretion of diagnostically important porphyrins (uro- and coproporphyrin), then total blood count, liver function tests (ALT and AST), blood sugar, cholesterin, serum iron and ferritin, and performed also urinanalysis and detection of possible anti-HCV antibodies. The laboratory examination was repeated in the patient subgroup after three months, urinary uro- and coproporphyrin excretion also in the the control group, for to have an opportunity to follow the dynamics of laboratory changes. All the patients were examined during their regular laboratory controls performed so as not to be bothered with repeated additional phlebotomies. We did not confirm in our patients suffering from breast tumor the results of other autors, suggesting the connection between tamoxifene-therapy and development of porphyria cutanea tarda. CONCLUSIONS: Isolated cases of PCT can be induced through the effect of various hepatotoxic factors. However, the influence of common porphyrinogenically acting noxious substances (alcohol, HCV virus or iron overload as a result of the HFE gene mutations) were not found in our patients.

[Non-melanocytic dischromias]. / Nemelaninové dyschromie.

Non-melanocytic hyperpigmentations (dyschromias), besides common melanotic ones, develop mostly due to the skin reaction to various chemicals and/or drugs of exogenous origin. They can be however also caused by some endogenously formed (evolving) intermediates of several metabolic diseases. The above mentioned factors are of toxic or allergic character and their clinical dermal manifestation often occurs as an allergic reaction after the exposition of the skin to solar radiation, or as a toxic contact reaction both on the skin and the mucosa. As a therapy of individual skin changes is difficult or impossible, the only treatment consists in reliable photoprotective measures.

[Urocanic acid and its role in the photoimmunomodulation process]. / Urokanová kyselina a její role v pochodech fotoimunomodulace.

Urocanic acid (UCA) is a metabolite of the amino acid histidine. It represents an important chromatophore in epidermis, which can absorb ultraviolet rays in UVB and UVA region and sequentially convert it from trans- to cis-isomer. Cis-isomer is not further degraded; it accumulates in the skin and is excreted with sweat and in shedding keratin scales. UCA has several important functions, the regulation of the homeostasis of the acidic cutaneous surface, the terminal differentiation of epidermal cells and namely the immunomodulatory role. As and immunomodulator UCA can suppress contact allergic reaction and the delayed hypersensitivity of the organism. It can affect reactions mediated by Th-lymphocytes, cytokine system, Langerhans cells, and by some neuropeptides. UCA is related to the development of non-pigmented skin tumors (basaliomas) and indirectly also to pigmented tumors. Cis-UCA can inhibit both the local and systemic resistance to infectious agents. In the immunomodulation some adductive compounds with another important cutaneous chromatophore DNA can participate.

Autoantibodies in sporadic porphyria cutanea tarda.

In an investigation of autoimmune antibodies using indirect immunofluorescence and Western blot analysis in a group of porphyria cutanea tarda patients we did not find any cytosolic antibodies potentially able to inhibit uroporphyrinogen decarboxylase. Furthermore, no known etiological factors were present in any of our patients. We therefore consider the development of the recently reported autoantibody with a molecular weight of 40 kDa a reaction to infection with the hepatitis C virus. The origin of mostly antinuclear antibodies against liver antigens (50, 45 and 56 kDa), detected in seven patients, was not identified and their etiopathogenetic implications remain unknown.

[Effect of ultraviolet radiation on the immune system and the effect of exogenous photochemoprotecive agents on the ultraviolet radiation induced immunosuppression]. / Vliv ultrafialového zárení na imunitní systém a pusobení zevních fotochemoprotektivních prostredku na UVR vyvolanou imunosupresi.

Ultraviolet radiation (UVR) of the UVB- and UVA-region suppresses immunity and in the dose relation manner it induces cutaneous or systemic state of immunotolerance. It was proved by the successful experimental transplantation of a tumor graft to the skin of UV-radiated animals and on the other side by unsuccessful attempts at skin contact-sensitization. UVR induces this condition either by direct inhibition of antigen-presentating function of the Langerhans cells, or indirectly by the stimulation of secretion of immunomodulatory cytokines in keratinocytes. UVR inhibits also functions of the natural killer cells in apoptotic and lytic cell-processes. It directly correlates with carcinogeneity in conditions of the dysbalance in developing expression of the tumor-suppressive gene p53, mutated by the irradiation and that of bcl-2 gene preventing apoptotic changes in somatic cells. Moreover, the immunosuppression is enhanced by the isomerization of urocanic acid and by the development of damaged DNA-photoproducts. Also various neuropeptides, namely propiomelanocortin, have their partial role in immunomodulation. Local photoprotective substances prevent UVR-penetration into the skin and thus block the immunosuppressive effects in relation to their chemical composition and to the SPF (solar photoprotective factor) value. Their effect depends on the width of the UVR-region blocked, which should include both the UVB and UVA band, and also on the concentration, chemical stability and on the method of their application.

Overt and hidden coinfection with hepatitis B and C viruses in chronic liver disease and porphyria cutanea tarda.

The aim of this study was to assess the rate of hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection ("the coinfection") in chronic liver disease (CLD) and to reveal overt and hidden HBV infection in patients with antibodies to HCV (anti-HCV). A total of 209 untreated patients (64 with chronic hepatitis B, 79 with chronic hepatitis C and 66 with porphyria cutanea tarda (PCT)) were screened for serological markers of HBV and HCV infection in serum by third generation enzyme-linked immunosorbent assay (ELISA) methods and for HBV DNA and HCV RNA in serum by polymerase chain reaction (PCR). The rate of the overt coinfection in chronic hepatitis B was very low (2/64, 3%). However, in chronic hepatitis C, the rate of the hidden coinfection with HBV was relatively high (19/79, 24%); these patients had higher alanine transaminase (ALT) and asparagine transaminase (AST) levels in serum and a more advanced liver disease. In PCT patients, the rates of HBV and HCV infections were the same, 21% (14/66). In the PCT patients infected with HBV or HCV, the rate of the coinfection was 33% (7/21). The PCT patients with the coinfection had a high serum ALT level and the worst histological picture in the liver. The hidden HBV infection was more frequent than the overt one. The possibility of the overt or hidden coinfection in CLD renders a detailed analysis of all serum samples for both viruses mandatory. Vaccination against HBV infection should be offered to anti-HCV-positive individuals as well as to PCT patients not showing antibodies to HBV (anti-HBV).

[Significance and prevalence of the C282Y gene mutation of primary hemochromatosis in the pathogenesis of pophyria cutanea tarda]. / Význam a prevalence mutace C282Y genu primární hemochromatózy (HFE) v patogenezi pozdní kozní porfyrie (PCT).

BACKGROUND: Hitherto studies on the ethiopathogenesis of porphyria cutanea tarda (PCT) show that the major pathogenic factor is iron ion, which acts via inhibition of the uroporphyrinogen decarboxylase. New speculations have appeared on the possible relation of this role of iron and the occurrence of mutation of the recently discovered gene of the hereditary hemochromatosis HFE, which may cause the iron overloading of the organism. Our paper describes prevalence of the C282Y gene mutation (HFE) together with the clinical and laboratory record in PCT patients. METHODS AND RESULTS: PCT was diagnosed mostly on the basis of clinical finding of actinic-traumatic vesicular dermatitis and the typical laboratory record of elevated higher-carboxylic porphyrines in urine and stool. Other laboratory methods tested the liver functions, plasma iron level and its binding capacity, ferritine level. All patient underwent routine haematological testing. Presence of antibodies against hepatitis C was also assayed (Elisa test 2nd generation, Sanofi Pasteur). In patients with prominent laboratory alterations showing possibility of the hepatic structural lesion, histology from the liver punctate was done. Frequency data of the C282Y gene mutation (HFE) in PCT patients was estimated on the basis of the genetic testing using PCR reaction of our own system. Group of PCT patients had 69 persons (63 patients with the sporadic form and 6 patients with familiar form of the disease). Hereditary haemochromatosis C282Y gene mutation (HH) was found in 15 patients, three of them were homozygotes and twelve heterozygotes (three heterozygotes had the familiar form of the disease). Nobody in this group was positive in the HIV antibody testing. In all porphyria patients with the presence of mutated gene who underwent liver biopsy, siderosis of different degrees was identified. In three patients neither the phenotypic observation nor the laboratory testing have shown haemochromatosis. Prevalence of C282Y gene mutation HFE in patients with porphyria cutanea tarda was studied. Such mutation was found in 15 persons (12 heterozygotes and 3 homozygotes) from the group of 69 tested patients (21.7%). Such frequency is significantly higher than in the control--nonporphyric--persons (10%). Patients were without clinical symptoms. Laboratory haematological changes, typical for HH, manifested in some of them only (elevated level of ferritine was found in 10 from 15 porphyria patients, elevated sideremia in one of them). Red blood cell counts were in both homo- and heterozygotes normal. Concurrence of the two porphyrinogenic factors--presence of gene mutation HFE and hepatitis C infection--was not proved. Antibodies against hepatitis C virus were not identified in any of the patients. Siderosis was found to be only a symptomatic sign, which was pronounced in different degree in all 9 porphyria patients with C282Y gene mutation who underwent liver biopsy. CONCLUSIONS: Frequency of C282Y gene mutation in our patients with porphyria cutanea tarda appears similar to that in other Middle European countries. It differs significantly from the frequency found in South European and North European countries (British).

[Porphyria cutanea tarda after antineoplastic drugs]. / Porphyria cutanea tarda po protinádorových lécích.

Chronic hepatic porphyria is in some cases a paraneoplasia, in others part of the neoplastic disease. Sometimes it is, on the other hand manifested during antineoplastic treatment. Its development may be associated with this therapy. The presented case-history describes a female patient with breast cancer treated with antineoplastic agents, who developed a sporadic form of late cutaneous porphyria with a classical clinical and laboratory picture. The authors contemplate on the possible porphyrinogenic effect of antineoplastic agents, in particular cyclophosphamide and tamoxifen, which in the latter preparation was not described so far.

[Chronic hepatic porphyria and hepatitis B and C virus infections]. / Chronická jaterní porfyrie a infekce virem hepatitidy B a C.

BACKGROUND: It is known that in patients with porphyria cutanea tarda (PCT) there is an increased prevalence of the hepatitis B virus (HBV) and the hepatitis C virus (HCV). The incidence of anti-HCV in PCT in our country is 21.7% in estimations by the second generation method, however, the incidence of HBV in PCT was not assessed so far. METHODS AND RESULTS: In 60 patients with PCT antigens and antibodies against HBV and HCV were assessed (by the anti-HCV third generation ELISA method) and in subjects with signs of HBV or HCV. HBV DNA and HCV RNA were assessed by the method of the polymerase chain reaction. PCT without detectable HBV or HCV infection was found in 45 subjects (68%). HBV infection only was confirmed in seven subjects (10.6%), however none of the patients had positive HBsAg in serum. All had only antibodies against HBV. HCV infection only was detected in seven patients (10.6%) and HBV and HCV co-infection also in seven patients (10.6%). In the group of patients with HBV and HCV co-infection there was not a single HBsAg positive subject. The mean ALT serum activity was significantly higher as compared with subjects with HBV or HCV infection only (p < 0.05) and the histological finding on liver biopsy was more serious. CONCLUSION: HBV (21%) and HCV (21%) infection participates significantly in the clinical picture of PCT. A special subgroup is formed by patients with PCT and HBV and HCV co-infection who have as a rule a higher ALT activity and more severe histological changes in the liver. The incidence of HBV and HCV infection in PCT in the Czech Republic is double as compared with Germany or Great Britain.

[Needle-like inclusions in liver cells are specific diagnostic signs of porphyria cutanea tarda]. / Jehlicovité inkluze v jaterních bunkách jsou specifickým diagnostickým znakem porphyria cutanea tarda.

Needle-shaped cytoplasmic liver cell inclusions are considered to represent a feature typical of porphyria cutanea tarda (PCT). Histological sections of 849 liver biopsies were stained with ferricyanide reduction reaction for demonstration of the inclusions. They were detected in 18 cases, and in all of them the diagnosis PCT was clinically and/or biochemically confirmed. In our group of patients, PCT was associated with alcoholic liver disease in three cases, with chronic hepatitis C in three cases and with hepatocellular carcinoma in two cases. In the liver with hepatocellular carcinoma, the inclusions were present only in non-neoplastic liver tissue, not in the tumourous tissue. No inclusions were found in the liver tissue of patients without clinical signs of PCT.

Screening of the gasoline lead load in school children in the Czech Republic.

The determination of erythrocyte zinc protoporphyrin (ZZP) was implemented as an indirect method for assessing lead levels in the blood in the follow-up of Czech children regarding the lead load due to automobile traffic. Simultaneously, basic indicators of the red blood picture were studied. A screening study was conducted with 2668 school-age children residing permanently in three different regions of our country. The differences in basic hematological indicators, i.e. number of erythrocytes, hemoglobin content, hematocrit and red blood cell volume, like the differences in zinc protoporphyrin in the peripheral blood, did not confirm the initial hypothesis on the relatively greater load of the Czech child population due to lead from exhaust fumes of combustion motors. Also, no regional differences were observed. The differences found in the abovementioned indicators reflect the influence of an overall environmental pollution rather than that of a toxic effect of lead.

[Erythrocyte zinc-protoporphyrin and characteristics of iron- deficiency anemia in parturient women and neonates]. / Erytrocytární zink-protoporfyrin a charakteristiky anémie z nedostatku zeleza u rodicek a novorozencu.

The authors examined the concentration of erythrocyte zinc-protoporphyrin (ZPP) by a haematofluorometric method in 175 women during delivery and the levels in their children. They assessed basic haematological indicators, the number of red cells, their volume, number of reticulocytes, haemoglobin concentration, haematocrit and sideraemia. All were average values within the physiological range in both examined groups. Statistical evaluation revealed a significant difference between mean values of sideraemia in mothers and children and mean values of the erythrocyte ZPP in both groups. The authors found also a medium significant correlation between ZPP and the erythrocyte volume and the erythrocyte volume and sideraemia. The correlation of different indicators was more marked after classification of the examined women and neonates into subgroups by haemoglobin concentrations and ZPP. In the subgroup of mothers with Hb values lower than 115 g/l a positive correlation was found with the number of reticulocytes red cell volume and sideraemia. In the subgroup of anaemic women (Hb concentration lower than 105 g/l) a negative correlation with the foetal sideraemia was found.