Studies on the genotoxic effect of beryllium chloride and the possible protective role of selenium/vitamins A, C and E

The genotoxic potential of beryllium chloride (BeCl₂) was evaluated in vivo in mice using different endpoints. Chromosomal aberrations in bone marrow cells and in spermatocytes as well as sperm abnormalities were determined in the tested mice. The protective role of an orally administered drug consisting of selenium and vitamins A, C and E (selenium–ACE) was also studied.For analysis of chromosomal aberrations, both single and repeated oral treatments for a period of 3 weeks were performed. The doses used were 93.75, 187.50, 375, and 750 mg BeCl₂/kg bw, which corresponds to 1/16, 1/8, 1/4, and 1/2 of the experimental LD₅₀.BeCl₂ induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells, with a dose– and time–response. The percentage of induced chromosomal aberrations was significantly reduced in all BeCl₂-treated groups after oral administration of selenium–ACE.Beryllium chloride also induced a significant increase in the percentage of abnormal sperm. This percentage reached values of 9.62 ± 0.32 and 5.56 ± 0.31 in mice treated with the highest test dose of BeCl₂ and with BeCl₂ + selenium–ACE, respectively, compared with 1.96 ± 0.14 for the control.In conclusion, the results demonstrate the genotoxic effect of beryllium chloride and confirm the protective role of selenium–ACE against the genotoxicity of beryllium chloride.     

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO⁻) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25–200 μM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50 μM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO⁻, were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations ≥50 μM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

Investigations on the mutagenicity of 1,4-dichlorobenzene and its main metabolite 2,5-dichlorophenol in vivo and in vitro

The genotoxic potential of 1,4-dichlorobenzene (1,4-DCB) has been extensively evaluated in vitro and in vivo. The majority of the studies demonstrated the absence of a genotoxic potential for 1,4-DCB. At variance are a bone marrow micronucleus test (MNT) after intraperitoneal (i.p.) treatment of NMRI mice [Mohtashamipur et al., Mutagenesis 2 (1987) 111–113] and a gene mutation assay on mouse lymphoma cells [McGregor et al., Environ. Mol. Mutagen. 12 (1988) 85–145]. Therefore, we investigated 1,4-DCB and its main metabolite 2,5-dichlorophenol (2,5-DCP) for both endpoints. In an MNT, male and female NMRI mice were treated orally with single doses of 2500 mg/kg 1,4-DCB and 1500 mg/kg 2,5-DCP, respectively. Smears were prepared 24, 48 and 72 h thereafter. No induction of micronuclei was detected for both compounds. Also under the conditions of Mohtashamipur et al. (1987), intraperitoneal treatments of male and female mice with 2 × 177.5 and 2 × 355 mg/kg 1,4-DCB failed to induce micronuclei. In addition, CHO/HPRT-gene mutation tests with 1,4-DCB and 2,5-DCP yielded negative results for both compounds with and without metabolic activation system. Therefore, 1,4-DCB and 2,5-DCP are considered to be non-mutagenic in these test systems.     

Regulation of resistance and susceptibility in wheat–powdery mildew pathosystem with exogenous cytokinins

Dose–response relationship between resistance of wheat seedlings (Triticum aestivum, cultivar Zarya) to Erysiphe graminis f. sp. tritici Marchal. (Syn. Blumeria graminis), a causal organism of wheat powdery mildew and exogenous zeatin has been investigated. Two-week-old seedlings were inoculated with the pathogen. Zeatin or zeatinriboside were added to the nutrient solution immediately after inoculation. The dose–response curve of cytokinin in the most cases was multiphasic, with peaks of increased susceptibility occurring at 0.25–1.5 and 1.5–9 μM cytokinin, separated by a region of increased resistance at 0.5–3 μM cytokinin. The change in mineral nutrition or simultaneous treatment with thidiazuron revealed alterations of the dose–response curve ranging from a curve with maximum of resistance to a curve with maximum of susceptibility. Both multiphase nature of dose–response and its variability were proposed as possible explanations for earlier observed discrepancies in experimental data on modification of disease resistance by cytokinins. A mathematical model for two metabolic processes with substrate inhibition connected in-series was suggested to explain the multiphase dose–response. In this model, the product of the first reaction was used as substrate for the second reaction. Numerical experiments showed the changes in the shape of dose–response curve with changes in parameters dependent of cytokinin metabolism.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology.Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

A comparison of genotoxicity between three common heterocyclic amines and acrylamide

Heterocyclic amines (HCAs), a group of genotoxic compounds formed during the heating of proteinaceous food items, have been known since the late 1970s. However, the genotoxic effect of these compounds in the low dose region has not yet been thoroughly studied. Here we used a sensitive flow cytometer-based micronucleus assay in mice to determine the frequency of micronucleated erythrocytes (fMPCE) of the three common HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), in the low dose region. We especially looked for any deviation from linearity of the dose–response curves. Male Balb/C mice were intra peritoneally injected with different doses of either PhIP (0–36 mg/kg b.w.), MeIQx (0–90 mg/kg b.w.) or IQ (0–40 mg/kg b.w.). In the case of PhIP, we found a significant dose–response relationship, while MeIQx and IQ did not display an increased fMPCE level. This flow cytometer method allows for determination of the DNA content of micronuclei. All three HCAs tested here yielded a low DNA content of micronuclei, indicating that they do not possess aneugenic effects. A comparison between the HCAs and acrylamide (AA), another heat induced genotoxic compound, revealed that the slope of the dose–response curve is about 10 times steeper for PhIP than AA. In spite of this, AA probably constitutes a higher human risk than HCAs since the intake is about a 100- to 1000-fold higher than the intake of HCAs.     

H2AX phosphorylation as a genotoxicity endpoint

The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4 h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60 μg/ml), the alkylating agent methyl methanesulfonate (1.3–65 μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10 μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500 μg/ml) and sodium chloride (0.058–580 μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay.These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.     

Investigating DNA damage in tannery workers occupationally exposed to trivalent chromium using comet assay

DNA damage of peripheral lymphocytes in 60 workers occupationally exposed to trivalent chromium [Cr(III)] in a tannery was studied using comet assay. The urinary and blood chromium levels were detected as a biomarker of internal exposure. The 90 subjects were divided into three groups: (i) exposure group I included 30 tannery workers highly exposed to chromium from tanning department; (ii) exposure group II included 30 tannery workers with moderate chromium exposure from finishing department; (iii) control group included 30 individuals without exposure to physical or chemical genotoxic agents. No significant difference was found among the three groups for age and smoking. The results showed that the medians of blood and urinary Cr of two exposure groups were significantly higher than those of control group (P < 0.01). And the medians of blood and urinary Cr of exposure group I were significantly higher than those of exposure group II (P < 0.05 or P < 0.01). The medians of mean tail length (MTL) of the three groups were 5.33 (2.90–8.50), 3.43 (2.31–8.29) and 2.04 (0.09–3.83) μm, respectively; The medians of mean tail moment (MTM) of the three groups were 6.28 (2.14–11.81), 3.41 (1.25–11.07) and 0.53 (0.13–3.29), respectively. The MTL and MTM of two exposure groups were significantly higher than those of control group (P < 0.01). The MTL and MTM of exposure group I were significantly higher than those of exposure group II (P < 0.01). The results of the present investigation suggest that occupational exposure to trivalent chromium can lead to a detectable DNA damage of human peripheral lymphocytes. Moreover, DNA damage was associated with chromium levels in blood. DNA damage may serve as a valuable effective biomarker and total chromium in blood may serve as a useful internal exposure biomarker in the population occupationally exposed to trivalent chromium.     

Phototherapy increases DNA damage in lymphocytes of hyperbilirubinemic neonates

Phototherapy is commonly used in the treatment of hyperbilirubinemia in newborns. No serious side effects related to phototherapy have been observed, but concerns regarding its potential to damage DNA have been expressed, based on animal or cell-culture studies. The aim of this study was to investigate, in neonates with hyperbilirubinemia, the possible relation between phototherapy and DNA damage. The study included 33 full-term newborns with non-physiological jaundice and 14 healthy newborns with physiological jaundice as controls. Phototherapy was performed with an array of six fluorescent lamps producing radiation with wavelengths of 480–520 nm at 12 μW/cm²/nm. DNA damage in lymphocytes was determined by use of the alkaline comet assay. The DNA damage increased significantly with the duration of phototherapy, as shown by measurements at 24, 48, and 72 h (P < 0.001). These findings indicate that phototherapy, widely used in neonatology units, increases DNA damage in newborns. It remains to be seen whether the genotoxic effect observed in the present study can cause any long-term health effect in phototherapy-treated infants in later life.     

Genotoxic effects of the pesticides Rubigan, Omite and Rovral in root-meristem cells of Crepis capillaris L.

Three pesticides have been studied for their genotoxicity by the use of assays in the plant Crepis capillaris, aimed at measuring chromosomal aberrations, micronuclei and sister chromosome exchange (SCE). The fungicides Rubigan 12 EC (fenarimol) and Rovral 25 Flo (iprodione) and the insecticide Omite 57 E (propargite) are all widely used nowadays. The aim of our study was to evaluate the genotoxic effects of these pesticides at concentrations corresponding to those applied in agricultural practice. In preliminary experiments we found that these concentrations do not influence cell proliferation and do not inhibit the growth of root meristems. In all experiments formulated commercial products were used. From the results we conclude that the three pesticides did not induce chromosomal aberrations as estimated by metaphase and anaphase analyses. They were also not capable to induce SCE. Rubigan did not induce micronucleus formation even at the highest concentration tested, but Omite and Rovral markedly increased micronucleus formation. The MN response depended on the sampling time and the concentration used, which showed a significant dose–response correlation (r = 0.978, P < 0.01 and r = 0.941, P < 0.01, respectively). A greater increase in micronucleus frequency was observed after Rovral treatment, where the highest concentration gave a response 8–10-fold above the negative control. Both pesticides induced high frequencies of lagging chromosomes, even after exposure to the lower test concentrations. The presence of lagging chromosomes is an indication of anti-microtubule activity of the pesticides tested. This effect was more strongly expressed after exposure to the two higher concentrations of Omite and Rovral. In this case a complete destruction of the mitotic spindle was observed, resulting in C-mitoses as well as in numerical aberrations—polyploidy and aneuploidy. The present findings suggest that Omite and Rovral at concentrations comparable to those used in practice can be regarded as potential aneugens.     

Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells

When chlorine is used as a disinfectant for drinking water it may react with organic materials present in or released by the water pipes and thus form by-products that may represent a genotoxic hazard. The aim of this study was to assess the potential genotoxicity and cytotoxicity of extracts of chlorinated drinking water supplied by local aquifers of two Italian towns, Plants 1 and 2, located in the sub-Alpine area and on the Po plain, respectively. The raw water fell within the legal limits with regards to its chemical and physical properties. Water from Plant 2 contained higher levels of total organics (TOC) and nitrate than water from Plant 1. Water was sampled at different points along the distribution networks to evaluate the influence of the system on the amount and quality of the by-products. Cytotoxic and genotoxic damage was assessed in freshly isolated human white blood cells (WBC) and Hep-G2 cells by use of the micronucleus (MN) test and the Comet assay to measure primary DNA damage. While they did not show significant cytotoxicity, all Plant 1 water concentrates induced short-time genotoxic effects on leukocytes at concentrations ≥1 Lequiv./mL. Plant 2 samples were able to induce cytotoxic effects in both Hep-G2 cells and leukocytes. Furthermore, although there was no significant increase in MN frequency, DNA migration was strongly increased both in human leukocytes (≥0.5 Lequiv./mL, 1 h treatment, water samples collected from all points) and in Hep-G2 cells (≥0.75 Lequiv./mL, 24 h treatment, tap water sampled at the nearest distribution point). The current use of these in vitro cytotoxicity/genotoxicity tests together with the normal chemical analyses could provide information to help water-works managers and health authorities evaluate drinking water quality and adopt strategies to reduce genotoxic compounds in tap water and prevent human exposure to these compounds.     

Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: determination of No-Observed-Genotoxic-Effect-Levels

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

The effect of early human contact and the separation method from the dam on responses of beef calves to humans

The objectives of this experiment were to evaluate the effect of early human contact and of the separation method from the dam on the future relationships of calves with humans, and to investigate the relationship between dam responses and calf responses. Thirty-three Salers calves aged 2–4 days old and reared outdoors were split into 3-week treatments balanced according to sex and birth dates. Group 1 (long separation from the dam and human contact: LS + H; n = 11) underwent 8 h of separation from the dam per day and 5 min of individual stroking; group 2 (short separation from the dam and human contact: SS + H; n = 11) underwent 1 h of separation and the same amount of human contact as LS + H calves. Group 3 (short separation from the dam and no human contact: SS − H; n = 11) was a control group undergoing the same duration of separation as SS + H but without stroking. At 3, 15 and 45 weeks of age, the calves were tested in a standard arena test (AT) where they were successively left alone (2 min), left with a stationary human (5 min), and left with a human approaching and touching them (2 min). At 15 and 45 weeks, the calves were also tested with the standard docility test (DT: test of restraint). The dams were also tested with DT 2 months before calving. Data analysis via Mann–Whitney tests and Spearman's correlations showed no significant effect of the duration calves were separated from their dams. Just after treatment at 3-week of age, calves given stroking (LS + H and SS + H) were more motionless and more willing to accept human contact (AT: touching) than control calves (SS − H, P < 0.01). At 45 weeks of age, calves given stroking spent also significantly more time (P < 0.05) motionless with the approaching human compared to non-stroked calves (SS − H), suggesting a persistent effect. However, this effect was not reproduced on the other behavioural criteria recorded (e.g., duration of human contact or docility score). In the different tests and at the different ages, the docility scores of the dams were significantly correlated (up to 0.7, P < 0.01) with behaviour towards humans shown by stroked calves but not non-stroked calves (SS − H). Our results suggest that additional human contact at early age, but not duration of the separation from the dam, could be beneficial for the human–animal relationship, but only for calves born to docile dams.     

Comparison of some novel polyculture and traditional monoculture vermicomposting reactors to decompose organic wastes

Efforts were made to evaluate the decomposition potentials of traditional monoculture and some novel polyculture vermireactors. Three earthworm species, i.e. Eisenia fetida (E. f.), Perionyx excavatus (P. ex.) and Lampito mauritii (L. m.), representing two different ecological categories: epigeic (E. fetida and P. excavatus) and anecic (L. mauritii), were used to design seven different vermireactors, i.e. Mono-(E. f.), Mono-(P. ex.), Mono-(L. m.), Poly-(E. f. + P. ex.), Poly-(P. ex. + L. m.), Poly-(E. f. + L. m.) and Poly-(E. f. + P. ex. + L. m.). The microbial load of vermireactors was evaluated through measuring dehydrogenises activities (DH-ase) and microbial biomass-N, while mineralization rate was measured in respect to changed level of some important nutrients in vermicomposted substrate. The vermicomposting caused decrease in pH (67.0–15.0%), organic C (46.1–28.4%) and C:N ratio (72.2–57.1%) and increase in total N (137.7–67.8%) as well as available P (107.9–16.9%) contents, at the end. The carbon and nitrogen mineralization rate showed the order: Poly-(E. f. + P. ex. + L. m.) > Poly-(E. f. + L. m.) > Poly-(P. ex. + L. m.) > Poly-(E. f. + P. ex.) > Mono-(E. f.) > Mono-(P. ex.) > Mono-(L. m.) for this study. The Poly-(E. f. + P. ex. + L. m.) vermireactor showed the maximum level of DH-ase activity 1926 ± 245 μg g⁻¹ substrate 24 h as well as microbial biomass-N 3059.1 ± 242.3 mg N g⁻¹ substrate, during experimentation. This study clearly suggests that burrowing earthworms in vermireactor not only promote the microbial colonization, but at the same time also accelerate the mineralization rate in decomposing waste. The polyculture vermicomposting, using burrowing earthworms with epigeics, could be more efficient than traditional monoculture vermireactors to decompose organic waste resources.     

Catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway

It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP⁺ and rotenone. Here, we further evaluated the effect of catalpol against Aβ_1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Aβ_1–42 was severed as cell model of Alzheimer's disease (AD) in vitro. By exposure to Aβ_1–42 (5 μM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Aβ_1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Aβ_1–42 through a mitochondrial-dependent caspase pathway.     

Genotoxic effects of PCB 52 and PCB 77 on cultured human peripheral lymphocytes

Polychlorinated biphenyls (PCBs) are known to be carcinogenic, but the mechanisms of this action are uncertain. Most, but not all, studies have concluded that PCBs are not directly mutagenic, and that much if not all of the carcinogenic activity resides in the fraction of the PCB mixture that contains congeners with dioxin-like activity. The present study was designed to determine genotoxic effects of an ortho-substituted, non-coplanar congener, 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), and a non-ortho-substituted coplanar congener with dioxin-like activity, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) on cultured human peripheral lymphocytes. DNA damage was assessed by use of the comet assay (alkaline single-cell gel electrophoresis). After cell cultures were prepared, test groups were treated with different concentrations of PCB 52 (0.2 and 1 μM) and PCB 77 (1 and 10 μM) for 1 h at 37 °C in a humidified carbon dioxide incubator, and compared to a DMSO vehicle control group. The cells were visually classified into four categories on the basis of extent of migration such as undamaged (UD), low damage (LD), moderate damage (MD) and high damage (HD). The highest concentration of PCBs 52 and 77 significantly increased DNA breakage in human lymphocytes (p < 0.001). Our results indicate that both the non-coplanar PCB 52 and coplanar PCB 77 cause DNA damage, and that the ortho-substituted congener was significantly more potent than the dioxin-like coplanar congener.     

Effect of Echinacea augustifolia extract on cell viability and differentiation in mammary epithelial cells

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system and activating biological property in different tissues. In this study we show the biological effect of Echinacea augustifolia extract on cell viability and cell differentiation in mammary epithelial cell lines. These effects have been observed in two different cell line derived from mouse (HC11) and bovine (BME-UV). Echinacea extract enhanced cell liability from 100 to 1000 ng/ml in association with growth factors, epidermal growth factor (EGF) or insulin, but also without EGF (p<0.05) up to 37% vs. control. This effect may be modulated by MAPK and Akt activation that Echinacea extract treatment increased and/or by a reduction of caspase 3 activity, showed a dose–response decrease after Echinacea treatment. Finally Echinacea extract was able to increase (p<0.05) at 100 ng/ml β-casein expression in association with PRL (5 μg/ml). These data demonstrate that Echinacea angustifolia extract can stimulate mammary epithelial cell physiology and may be considered a candidate to support mammary gland activity during a mammogenetic and lactogenetic state.     

Antioxidative response of Lemna minor plants exposed to thallium(I)-acetate

The physiological effects of thallium(I)-acetate on the duckweed Lemna minor after 1-, 4-, 7- and 14-d exposure were analyzed. High bioaccumulation of Tl (221 mg kg⁻¹ dry wt at 2.0 μM Tl-acetate) caused an inhibition of plant growth. After 14-d exposure, 0.2, 0.5, 1.0 and 2.0 μM Tl-acetate reduced the frond-number growth rate by 21.1%, 39.4%, 66% and 83.1%, respectively. Tl-acetate also induced a modulation of the antioxidative response by depleting the ascorbate content and affecting the antioxidative enzymes activities. Superoxide dismutase showed a continuous increase of activity (31–67%) after Tl-acetate exposure. Other antioxidative enzymes displayed a biphasic response to both the concentration and the exposure period. Exposure up to 7 d decreased the catalase activity (up to 40%) in plants treated with higher Tl-acetate concentrations. In contrast, 14-d exposure increased the activity of the enzyme (≥90%). Short-term exposure increased ascorbate peroxidase activity (13–41%), except in plants exposed to the highest Tl-acetate concentration. However, 14-d exposure decreased the enzyme activity at all concentrations tested (38–60%). Although pyrogallol peroxidase activity increased (up to 26%) during 4-d exposure, longer exposures to the highest two concentrations decreased the activity of the enzyme (25–48%). In general, short-term exposure to Tl-acetate activated the antioxidant capacity, which resulted in recovery of the frond-number growth rates in Tl-treated plants. In spite of the activation of the antioxidative response during short-term exposure, higher Tl-acetate concentrations increased the hydrogen peroxide level (up to 45%) and induced marked oxidative damage to lipids, proteins and DNA. Longer exposure induced a decline of the antioxidative response, and plants showed the symptoms of oxidative damage even at lower Tl-acetate concentrations. The genotoxic effect was evaluated by an alkaline version of the cellular and acellular Comet assay, which revealed an indirect genotoxic effect of Tl-acetate, suggesting oxidatively induced damage to DNA.     

Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.