Validation and application of sub-2 µm core-shell UHPLC-UV-ESI-Orbitrap MS for identification and quantification of ß-carotene and selected cleavage products with preceding solid-phase extraction.

A validated ultrahigh-performance liquid chromatography method using 1.7 µm core-shell particles is presented for the identification and quantification of ß-carotene (BC) and related cleavage products (CPs) in primary cell culture media. Besides BC, apo-4'-, apo-8'-, apo-10'-, and apo-12'-carotenals, as well as 5,6-epoxy-ß-carotene, were selected as target analytes. Detection was performed via an 80-Hz diode array detector and an electrospray ionization-linear quadrupole ion trap-Orbitrap XL mass spectrometer, both hyphenated in series. Total analysis time was below 6 min with peak widths <12 s. Addition of trifluoroacetic acid and tetrahydrofuran to the mobile phase allowed for the mass spectrometric detection of BC and related CPs and reduced peak tailing due to improved solubility of hydrophobic analytes. Intra-day and inter-day precision for UV and mass spectrometric detection were ≤1.5 % for retention times and ≤5.1 % for peak areas. Instrumental linearity was confirmed by Mandel's fitting test between 0.25 (or 1.00 µg/mL) and 5.00 µg/mL for UV detection. The higher sensitivity of mass spectrometric detection allowed for the coverage of three concentration domains between 0.025 and 5.00 µg/mL in linearity testing. Homoscedasticity was confirmed between 0.10 and 5.00 µg/mL for Orbitrap XL MS. The limits of quantification were between 52.6 and 889.4 ng/mL for UV detection and between 19.3 and 102.4 ng/L for mass spectrometric detection. Offline solid-phase extraction from culture media fortified with BC and CPs provided intra- and inter-day recoveries between 65.8 and 102.4 % with coefficients of variation ≤6.2 %. Primary rat hepatocyte cultures treated with BC and subjected to different oxidative stress conditions contained 5,6-epoxy-BC and apo-4'-carotenal besides residual BC. Apparently, 5,6-epoxy-BC was formed in the medium via autoxidation of BC by ambient oxygen.

Solid-phase extraction and GC-MS analysis of potentially genotoxic cleavage products of ß-carotene in primary cell cultures.

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of ß-carotene in cell culture media has been developed. Target CPs comprised ß-ionone (ß-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 µg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for ß-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method.

N-nitrosodiethylamine genotoxicity evaluation: a cytochrome P450 induction study in rat hepatocytes.

In rats, N-nitrosodiethylamine (NDEA) induces tumors mainly in the liver. This could be because various enzymes are responsible for the metabolic activation of NDEA, besides the hepatic NDEA metabolizing enzyme, CYP2E1. We examined NDEA genotoxicity and cytotoxicity in primary cultures of female rat hepatocytes; we also looked at how it affected CYP mRNA expression. Single incubation with 0.9% NaCl resulted in a mean of 0.2% apoptotic cells, which doubled with 105 µg NDEA/mL. The frequency of necrosis with NDEA treatment was also doubled. Besides the cytotoxic effects, there was also a 4-fold decrease in mitotic index and a 3-fold decrease in the percentage of cells with micronuclei. A significant increase in micronucleus cells when hepatocytes were incubated with 2.1 µg NDEA/mL suggests that DNA repair was inactive. The chromosomal aberration evaluation revealed a discrete dose-response curve. Treatment with NDEA induced increases in CYP mRNA: CYP2B2 (1.8 times) and CYP2E1 (1.6 times) with non-cytotoxic NDEA concentrations (0.21-21 µg/mL). CYP2B1 mRNA levels decreased at 0.21 µg NDEA/mL (2.5-fold), while CYP4A3 mRNA decreased 1.3-fold. NDEA treatment at 2.1 µg/ mL induced a 1.9-fold increase in CYP3A1 mRNA. Understanding the cumulative effects in target cells during precarcinogenesis is crucial to understanding the mode of action of potential carcinogens and in order to develop comprehensive chemical toxicity profiles.

Ferritin and FasL (CD95L) mediate density dependent apoptosis in primary rat hepatocytes.

Based on a recent description of an apoptosis stimulating property for hepatocyte derived isoferritins, this investigation demonstrates that ferritin, released in vitro from hepatocytes substantially contributes to density dependent apoptosis in primary hepatocytes and is significantly (P < or = 0.05) inhibited by anti-H-ferritin antibody rH02. Furthermore, total protein release and albumin secretion rapidly decline in a time and density dependent mode under serum-free conditions, whereas ferritin secretion, which is upregulated at initial stages of primary culture is not affected by cell density. Supplementation with dexamethasone (DEX) or proliferative stimulation by epidermal growth factor (EGF) and insulin strongly suppresses density dependent apoptosis. Both regimens have previously been shown to inhibit isoferritin mediated apoptosis in hepatocytes, most likely by interrupting proapotitc mitochondrial signalling. Finally, FasL/Fas also participates in density dependent apoptosis, since apoptosis is significantly (P < or = 0.005) reduced in high density cultures supplemented with an anti-FasL antibody. This antibody has also been shown to neutralise ferritin mediated apoptosis in primary hepatocytes, suggesting a linkage of ferritin and Fas in density dependent apoptosis. In conclusion, ferritin contributes to apoptosis in primary hepatocytes in an autocrine, density dependent mode, involving Fas stimulation and proapoptotic mitochondrial signalling. With respect to liver physiology, these findings may indicate that ferritin plays a yet unrecognised role as an acute phase signalling molecule in early stages of tissue repair and liver regeneration, and may also be responsible for the limited ability to propagate human hepatocytes in culture and the limited expansion of donor cells in the recipient liver upon cell transplantation.

Evaluation of the toxicological properties of ground- and surface-water samples from the Aral Sea Basin.

In order to determine whether there is a potential health risk associated with the water supply in the Aral Sea Basin, ground- and surface-water samples were collected in and around Aralsk and from the Aral Sea in 2002. Water samples from Akchi, a small town close to Almaty, served as controls. Bioassays with different toxicological endpoints were employed to assess the general toxicological status. Additionally, the samples were analysed for microbial contamination. The samples were tested in the primary hepatocyte assay for their potential to induce micronuclei and chromosomal aberrations as cumulative indicators for genotoxicity. In parallel, the effects on cell proliferation evidenced by mitotic index and cytotoxicity such as the appearance of necrotic and apoptotic cells, were determined. Furthermore, samples were examined using the Microtox assay for general toxicity. Chemical analysis according to European regulations was performed and soil and water samples were analysed for DDT and DDE. The results obtained indicated no increased cyto- or genotoxic potential of the water samples, nor levels of DDT or DDE exceeding the thresholds levels suggested by WHO. Our data therefore do not support the hypothesis that the contamination of the drinking water in and around Aralsk is responsible for the health effects previously described such as increased rates of liver disease and in particular liver cancer. Microbiological analysis, however, revealed the presence of contamination in most samples analysed.

Effects of aqueous extracts of medicinal plants on MNNG-treated rat hepatocytes in primary cultures.

Aqueous extracts of Nigella sativa (Ranunculaceae) (Ns), Teucrium polium (Labiatae) (Tp) and Trigonella foenum-graecum (Fabaceae) (Tf) have been traditionally used to treat inflammations, liver disorders, and arthritis. Experimentally, it has been demonstrated that these herbs possess antioxidant, anti-inflammatory and hepatoprotective properties. To evaluate their in vitro toxicological properties and potential antimutagenic effects aqueous extracts of the three plants were tested in primary rat hepatocyte cultures against N-methyl-N'-nitro-N-nitrosoguanidine. The extracts were applied before, during and after application of MNNG to discriminate between different mechanisms of action. Tp itself significantly increased apoptosis, but in the combined treatment with MNNG significantly reduced it. Post-treatment with Ns or combined treatment with Tf significantly reduced the percentages of necrotic cells. The three plant extracts themselves significantly increased the frequency of chromosomal aberrations. Summarizing, our results suggest that aqueous extracts of the three herbs have neither cytoprotective nor antimutagenic activity, instead there is evidence for a mutagenic potential.

Cyto- and genotoxic potential of beta-carotene and cleavage products under oxidative stress.

Free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria, a finding which could provide an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators for the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo-8'-beta-carotenal (apo-8') and beta-carotene in the primary rat hepatocyte assay in the presence and absence of oxidative stress provided by hypoxia/reoxygenation (Hy/re). The endpoints tested were: the mitotic indices, the percentages of necrotic and apoptotic cells, micronucleated cells (MN), chromosomal aberrations (CA) and sister chromatid exchanges (SCE). The results obtained indicate a genotoxic potential of both CP and apo-8' already in the concentration range of 100 nM and 1 microM, i.e. at physiologically relevant levels of beta-carotene and beta-carotene breakdown products. In contrast, no significant cytotoxic effects of these substances were observed, nor did beta-carotene induce significant cytotoxic or genotoxic effects at concentrations ranging from 0.01 up to 10 microM. However, when beta-carotene is supplemented during oxidative stress induced by hypoxia/reoxygenation, a dose-dependent increase of CP is observed accompanied by increasing genotoxicity. Furthermore, when beta-carotene cleavage products were supplied during oxidative stress significant additional increases of genotoxic effects were observed, the additional increases indicating an additive effect of both exposures. Summarizing, these results provide strong evidence that beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the beta-CArotene and RETinol Efficacy (CARET) Trial.

Analytical tools for the analysis of ß-carotene and its degradation products.

ß-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on ß-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with ß-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of ß-carotene. Their generation originates in the polyene structure of ß-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs). Due to their instability and hydrophobicity, the handling of standards and real samples containing ß-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.

An in vitro micronucleus assay for determining the radiosensitivity of hepatocytes.

An in vitro micronucleus assay was developed for primary cultures of rat hepatocytes and utilized to determine the oxygen enhancement ratio (OER). Freshly isolated Fischer 344 female rat hepatocytes were irradiated in suspension either in air or in anoxia, cultured for 60 h to allow for the maximum expression of micronuclei, fixed with methanol-glacial acetic acid, stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI), and scored with a fluorescent microscope for the presence of micronuclei. The percentage of cells with micronuclei increased linearly with dose, and the slopes of the relationships were 0.044 +/- 0.001 Gy-1 and 0.015 +/- 0.001 Gy-1 in air and anoxia, respectively. The calculated OER of 2.9 +/- 0.5 is similar to that previously obtained for hepatocyte cell survival. Our data demonstrate that this in vitro hepatocyte micronucleus assay is a rapid and sensitive method to further investigate those factors which influence the radiosensitivity of hepatocytes.

Adaptive response and dose-response plateaus for initiation in a state-vector model of carcinogenesis.

PURPOSE: To investigate whether it is possible to explain dose-response plateaus for in-vitro X-ray irradiation of different cell lines with radioprotective mechanisms such as radiologically induced expression of scavengers and repair enzymes. MATERIALS AND METHODS: A biomathematical model was developed based on a previous state-vector model. New features of the model are a mathematical description of enhanced repair and radical scavenging as a result of irradiation. RESULTS: The model produces a plateau in the dose-response for in-vitro tranformations between 0.5 and 1 Gy and for chromosome aberrations and it predicts an inverse-fractionation effect within a selected range of doses. CONCLUSIONS: Adaptive response mechanisms within a state-vector model provide a coherent explanation of the dose-response characteristics for in-vitro transformations and chromosomal aberrations. These results suggest the need for new experimental studies described in the paper.

Significance of cell-cycle delay, multiple initiation pathways, misrepair and replication errors in a model of radiobiological effects.

PURPOSE: To advance a biomathematical model of radiocarcinogenesis by describing multiple pathways for initiation, a radiologically induced cell-cycle delay, misrepair and spontaneous DNA damages caused by replication. It was investigated whether the incorporation of these biological features would improve the fit of the model to data showing plateaus in in vitro irradiations of different cell lines and whether the fit parameters were then more biologically realistic. MATERIALS AND METHODS: A biomathematical submodel was developed based on a previous State-Vector Model that mathematically described enhanced DNA repair and radical scavenging following irradiation. RESULTS: With the two initiation pathways and cell-cycle delay, the simulations better explained the mouse data but not the rat data, and for both data sets the fit parameters were biologically more realistic than previously assumed. Inclusion of misrepair and replicational errors did not significantly affect the fit. CONCLUSIONS: A plateau in the dose-effect relationship for in vitro irradiation of different cell lines can be explained by radioprotective mechanisms. The plateau-type dose-response relationships point to a non-linear dose- effect relationship at low doses and indicate that linear extrapolation from moderate (or high) to low doses may not be justified for in vitro studies of these cell lines.

Genotoxic effects of selected peroxisome proliferators.

Peroxisome proliferators, a class of structurally dissimilar chemicals including hypolipidemic drugs and industrial plasticizers, have been shown to be associated with hepatocarcinogenesis although an initiating effect could not yet be demonstrated in the cell systems utilized. For this reason the genotoxic potential of the peroxisome proliferators nafenopin, ciprofibrate and di(2-ethylhexyl)adipate (DEHA) was determined in primary cultures of adult rat hepatocytes. To further test if these compounds are genotoxic per se or the genotoxic effect is due to peroxisome proliferation, the cultures were exposed for 3 and 51 h. Treatment for 3 h with the hypolipidemic drugs nafenopin and ciprofibrate induced statistically significant increases of SCE at concentrations > or = 30 and 100 microM respectively. At higher concentrations statistically significant increases of chromosomal aberrations (nafenopin: 100 microM; ciprofibrate: > or = 100 microM) and micronuclei (ciprofibrate: > or = 250 microM) were also found. The presence of peroxisome proliferators in the media until harvesting (51 h) did not significantly alter the dose response of SCE, micronuclei and chromosomal aberration induction by ciprofibrate, while long-term exposure to nafenopin resulted in statistically significant increases of chromosomal aberrations and micronuclei at concentrations > or = 30 microM. The differences were statistically significant at 30 and 100 microM for micronuclei, and at 30 microM for chromosomal aberrations. Neither short- nor long-term exposure to DEHA produced a significant genotoxic effect up to 200 microM. The peroxisome proliferators tested were not cytotoxic at any concentration, as determined by mitotic index. These results clearly demonstrate that the peroxisome proliferators nafenopin and ciprofibrate can cause genotoxic effects in primary cultures of adult rat hepatocytes. The comparison of short- and long-term exposure does not suggest a strong correlation between the induction of peroxisome proliferation and genotoxicity, since long-term exposure did not significantly alter the dose response and--except for nafenopin--the extent of the genotoxic effects.

The primary rat hepatocyte micronucleus assay: general features.

Recent advances in cell culture have allowed proliferation of primary rat hepatocytes enabling the analysis of different cytogenetic endpoints such as sister chromatid exchanges (SCE), chromosomal aberrations and micronuclei. The latter are of particular interest as preparation, staining and analysis is less time-consuming than the analysis of chromosomal aberrations and SCE what makes micronuclei an attractive short-term assay. This paper gives (1) a summary of the specific features of primary hepatocytes including ploidy, nuclearity and multipolar mitoses, (2) a summary of the culture conditions for proliferation and the proliferation kinetics, (3) an experimentally based interpretation of the comparatively high background levels of cells with micronuclei, and (4) an experiment-based discussion of approaches to circumvent the major disadvantage: the cytochalasin B method cannot be applied due to the high percentage of binucleated cells.

Possible mutagens derived from lipids and lipid precursors.

Free radicals can initiate the oxidative decomposition of cellular membranes by lipid peroxidation. In this process a great variety of reactive aldehydes are produced intracellularly. Some of them, such as 4-hydroxynonenal or malonaldehyde, are biologically very active and might be involved in free radical-mediated DNA damage. A short review of the effects of aldehydic lipid peroxidation products on isolated DNA, their genotoxic effect in prokaryotes and eukaryotes and their in vivo carcinogenicity is given. Additionally own experiments on cytotoxic and genotoxic effects of 4-hydroxynonenal, 2-nonenal and nonanal in primary cultures of rat hepatocytes are reported. 4-Hydroxynonenal was highly cytotoxic at 100 microM, at subcytotoxic concentrations of 0.1-10 microM 4-hydroxynonenal increased the frequency of micronuclei, chromosomal aberrations and sister-chromatid exchange. 2-Nonenal and nonanal were not cytotoxic at 100 microM, the maximum dose tested. At 100 microM 2-nonenal led to a slight increase in micronuclei; chromosomal aberrations were not significantly altered. Nonanal had no detectable genotoxic effects. The level of endogenous 4-hydroxynonenal in tissues is in the range of 0.1-3.0 microM and can increase to 10 microM in conditions of oxidative stress; such levels appear to be sufficiently high to produce DNA damages, whether such damages are transient or irreversible is not known.

Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes.

4-Hydroxynonenal (HNE), one of the major products of lipid peroxidation, has been demonstrated to induce genotoxic effects in the micromolar range. HNE has too structural domains, a lipophilic tail and a polar head with three functional groups: the aldehyde and hydroxy groups and the trans CC double bond. To evaluate their relative importance, the genotoxic effects of HNE were compared with those of the homologous aldehydes 4-hydroxyhexenal and 4-hydroxyundecenal (different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonenal (lacking the OH group) and nonanal (lacking the OH group and the trans CC double bond). This investigation was carried out on primary cultures of adult rat hepatocytes in order to further determine the influence of biotransformation- and/or detoxification reactions. A 3-h treatment with HNE induces statistically significant levels of SCE at concentrations > or = 0.1 microM, micronuclei at concentrations > or = 1 microM and chromosomal aberrations at a concentration of 10 microM. Compared to HNE the homologous aldehydes induced a significant genotoxic effect at higher concentrations. Statistically significant increases in SCE frequency were obtained at concentrations > or = 1 microM for 4-hydroxyundecenal and at a concentration of 10 microM for 4-hydroxyhexenal. The induction of chromosomal aberrations was significantly elevated at concentrations of > or = 10 microM and 10 microM for 4-hydroxyhexenal and 4-hydroxyundecenal, respectively. Except for a 4-hydroxyhexenal concentration of 1 microM, both aldehydes did not induce statistically significant levels of micronuclei. The HNE analogous aldehydes 2-trans-nonenal and nonanal induced statistically significant frequencies of SCE at concentrations of > or = 1 microM (nonanal) and > or = 10 microM (2-trans-nonenal). No significant induction of chromosomal aberrations or micronuclei could be demonstrated. The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways. (1) The length of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytotoxic potential. (2) The lack of the OH group (2-trans-nonenal) reduces the SCE-inducing potential of the aldehyde shifting the dose-effect curve to higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxic and genotoxic effects of 4-hydroxynonenal in cerebral endothelial cells.

Oxygen free radicals are produced in the central nervous system (CNS) as a consequence of normal physiological metabolic reactions of neuronal cells, but there is evidence accumulating that they are also implicated in the processes leading to a number of pathological changes in the brain. A general mechanism whereby oxygen free radicals induce tissue damage is lipid peroxidation (LPO), which generates a large variety of water-soluble carbonyl compounds. Due to their high reactivity, we focused our investigations on 4-hydroxyalkenals, in particular on 4-hydroxynonenal (HNE), the major 4-hydroxyalkenal. Two phenotypes of cerebral endothelial cells (cECs) were treated with various concentrations of 4-hydroxynonenal and the cyto- and genotoxic effects studied. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Three hours of incubation with HNE induced significantly elevated levels of chromosomal aberrations at concentrations > or = 1 microM and micronuclei at concentrations > or = 10 microM in both cEC phenotypes, compared to the controls. Cytotoxicity was observed at a concentration of 50 microM HNE and was significantly higher in the elongated and spindle-shaped cEC phenotype (type II) than in the epithelial cEC phenotype (type I). The results indicate that cECs are affected by HNE even at low concentrations with minor differences between the two cEC phenotypes.

Genotoxic effects of three Fusarium mycotoxins, fumonisin B1, moniliformin and vomitoxin in bacteria and in primary cultures of rat hepatocytes.

The genotoxic effects of three widespread Fusarium toxins, vomitoxin (VOM), moniliformin (MON) and fumonisin B1 (FB1) were investigated in bacterial tests and in micronucleus (MN) and chromosomal aberration (CA) assays with primary rat hepatocytes. All three toxins were devoid of activity in gene mutation assays with Salmonella typhimurium strains TA98 and TA100 and in SOS chromotests with E. coli strain PQ37 in the presence and absence of metabolic activation. FB1 and VOM gave negative results in differential DNA repair assays with E. coli K-12 strains (343/753, uvrB/recA and 343/765, uvr+/rec+); with MON, a marginal effect was seen in the absence of metabolic activation mix at relatively high concentrations (> or = 55 micrograms/ml). In metabolically competent rat hepatocytes stimulated to proliferate with EGF and subphysiological Ca2+ concentrations, a decrease of cell division was observed with all three toxins at concentrations > or = 10 micrograms/ml, VOM was strongly cytotoxic at 100 micrograms/ml. All three mycotoxins caused moderate increases of the MN frequencies at low concentrations (< or = 1 microgram/ml), but no clear dose-response effects were seen and at higher exposure levels the MN frequencies declined. In the CA experiments with hepatocytes, pronounced dose-dependent effects were observed with all three toxins. MON caused a 9-fold increase over the spontaneous background level after exposure of the cells to 1 microgram/ml for 3 h, with FB1 and VOM, the increases were 6- to 7-fold under identical experimental conditions. This is the first report on clastogenic effects of VOM and FB1 in mammalian cells, with MON induction of CAs in V-79 cells has been described earlier. Since all three mycotoxins caused CAs at very low concentration levels in liver cells in vitro, it is possible that such effects may also occur in humans and mammals upon consumption of Fusarium-infected cereals.

Chromosomal aberrations of blood lymphocytes induced in vitro by radon-222 daughter alpha-irradiation.

Blood samples were irradiated in vitro with alpha-rays emitted from short-lived radon decay products dissolved in the culture medium at doses between 0.03 and 41.4 mGy. The data were collected from experiments conducted during the period 1984-1992 and comprise a total of about 64000 scored metaphases. For statistical reasons, only 60,022 metaphases were used for the subsequent analysis. The results for total chromosome aberrations and dicentrics indicate a linear dose dependence in the dose range above about 10 mGy, consistent with other experimental observations. At doses below about 10 mGy, aberration frequencies cannot be linearly extrapolated from higher doses, suggesting that there is no dependence on dose within a certain low-dose range. In addition, a statistically significant minimum has been observed at a dose of about 0.03 mGy, which is consistently lower than the related control values. The behavior of the aberration frequencies in the low-dose region seems to be influenced by the control values, which also depend on the environmental radiation burdens to the donors before blood sampling and thus were significantly affected by the Chernobyl fallout.

Cyto- and genotoxic potential of the photosensitizer Photosan 3 in the absence of light.

In the present investigation, cultures of rat hepatocytes were treated with Photosan 3 (PS 3) and examined for possible mutagenic effects in the absence of light. Hepatocytes are particularly useful in genotoxicity studies because of their capacity to metabolize a large variety of promutagenic/procarcinogenic compounds. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Three hours of incubation with PS 3 (at concentrations of 0.1, 1, 10, and 100 micrograms ml-1) induced significantly elevated levels of chromosomal aberrations and micronuclei already at the lowest concentration of 0.1 micrograms ml-1 compared with the controls. A concentration of 100 micrograms ml-1 PS 3 appeared to be cytotoxic: no mitotic figures could be detected.

Does delta-aminolaevulinic acid induce genotoxic effects?

5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway of haem. The presence of exogenous ALA bypasses the feedback control and may induce the accumulation of PpIX. Since haem-containing enzymes are essential for energy metabolism, every nucleated cell in the body must have at least a minimal capacity to synthesize PpIX. Photodynamic therapy (PDT), which is the treatment of malignant lesions with light following the administration of a tumour-localizing photosensitizer, leads to oxidative damage, including the formation of genotoxic membrane degradation products via lipid peroxidation. In addition, it has been demonstrated that ALA itself can form the reactive oxygen species Ox.-, H2O2 and OH. by auto-oxidation, suggesting that it could potentially induce DNA damage. Therefore cultures of rat hepatocytes, which have been demonstrated to be very sensitive indicators for genotoxic effects induced by the lipid peroxidation product 4-hydroxynonenal and analogous aldehydes, were examined for possible mutagenic effects after treatment with ALA in the absence of light. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Compared with the controls, significantly elevated levels of chromosomal aberrations and micronuclei were observed at concentrations of 1 microgram ml-1 or greater. Chromosomal aberrations and micronuclei were found to increase up to a concentration of 100 micrograms ml-1 ALA. While micronuclei decrease at higher concentrations, chromosomal aberrations remain at the same level. The kinetics of PpIX formation after induction with 100 and 1000 micrograms ml-1 ALA appear to be the same for both concentrations, suggesting that the induction of chromosomal aberrations may be due to PpIX.