Antigenotoxic and antimutagenic effects of lignin derivative BP-C2 against dioxidine and cyclophosphamide in vivo in murine cells.

The study investigated antigenotoxic and antimutagenic activity of novel lignin-derived polyphenolic composition (BP-C2) with ammonium molybdate towards cyclophosphamide and dioxidine in the bone marrow, blood and liver cells of BALB/c mice. BP-C2 was given to mice via gavage at 60, 80 and 120 mg/kg once 1 h before single intraperitoneal injection of a genotoxic agent. 1.5 h and 3 h after dioxidine or cyclophosphamide injection, respectively, cellular suspensions were obtained from mice and assessed with the comet test and cytogenetic analysis of bone marrow cells. It was observed that antigenotoxic activity of BP-C2 against DNA damage induced by dioxidine, a prooxidant genotoxic agent, in the bone marrow, liver and blood cells of mice in vivo was more pronounced at 60 and 80 mg/kg than at 120 mg/kg. When cyclophosphamide was used to induce a DNA damage, the genoprotective effect of BP-C2 was observed in bone marrow, liver and blood cells at 60 mg/kg dose but the effect was not significant at 80 mg/kg. When co-administered with 120 mg/kg BP-C2, cyclophosphamide induced a higher level of DNA damage in liver cells, but its genotoxic effect in bone marrow and blood cells was the same as when it was administered alone. When assessing the effect of BP-C2 on chromosomal aberrations induced by cyclophosphamide and dioxidine in bone marrow cells, it was revealed that all three tested doses of BP-C2 significantly decreased the number of cells with chromosome abnormalities. Thus, BP-C2 has a pronounced antimutagenic and genoprotective effects.

Comet assay on one- and two-cell mouse embryos.

DNA damage quantified as the comet tail length was assessed using in vitro and in vivo comet assay on one- and two-cell mouse embryos obtained by natural mating. The use of a protocol with three layers of agarose reduces the embryo loss and makes it possible to study a small number of embryos. A significantly lower level of basal, but not induced DNA damage was found in embryos with cleaved zona pellucida compared to embryos with intact zona pellucida. There were no significant differences in the length of the comet's tail between embryos lysed in different lysis solutions, both in cases of basal and induced DNA damage. A significant increase in the comet tail length was detected in one-cell embryos of mice treated with methyl methanesulfonate and etoposide compared to the control. The data show that DNA damage induced in maternal germ cells persists, which can be detected in embryos using the comet assay.

Genotoxicity of cationic lipopeptide nanoparticles.

The genotoxicity of cationic lipopeptide nanoparticles (cLPNPs) was evaluated in vivo and in vitro comet assay and the in vivo chromosome aberrations test. In vitro comet assay, human blood cells were exposed to cLPNPs at the concentration of 2.5, 5, 10, 20, 40 and 100 µg/mL. Significant DNA damage was observed after 1 h exposure, but no effects were detected after 3 h. In vivo, cLPNPs were administered in single or five daily injection doses at 8, 20 and 40 mg/kg of body weight by subcutaneous injection to male mice. The cLPNPs caused DNA damage in the liver, lung and kidney, but not in the spleen. The kidney was more prone to genotoxic effects that persisted from 24 h to 14d after a single injection of cLPNPs. No statistically significant increase in the percentage of cells with chromosomal aberrations above the vehicle control was observed in mice bone marrow after a single or repeated injection of cLPNPs. In summary, cLPNPs shown to be genotoxic both in vivo and in vitro. The results suggest the importance of the use of highly sensitive methods, such as the comet assay, in order to determine the full genotoxic potential of nanoparticles.

Alterations in the nigrostriatal system following conditional inactivation of α-synuclein in neurons of adult and aging mice.

The etiology and pathogenesis of Parkinson's disease (PD) are tightly linked to the gain-of-function of α-synuclein. However, gradual accumulation of α-synuclein aggregates in dopaminergic neurons of substantia nigra pars compacta (SNpc) leads to the depletion of the functional pool of soluble α-synuclein, and therefore, creates loss-of-function conditions, particularly in presynaptic terminals of these neurons. Studies of how this late-onset depletion of a protein involved in many important steps of neurotransmission contributes to PD progression and particularly, to worsening the nigrostriatal pathology at late stages of the disease are limited and obtained data, are controversial. Recently, we produced a mouse line for conditional knockout of the gene encoding α-synuclein, and here we used its tamoxifen-inducible pan-neuronal inactivation to study consequences of the adult-onset (from the age of 6 months) and late-onset (from the age of 12 months) α-synuclein depletion to the nigrostriatal system. No significant changes of animal balance/coordination, the number of dopaminergic neurons in the SNpc and the content of dopamine and its metabolites in the striatum were observed after adult-onset α-synuclein depletion, but in aging (18-month-old) late-onset depleted mice we found a significant reduction of major dopamine metabolites without changes to the content of dopamine itself. Our data suggest that this might be caused, at least partially, by reduced expression of aldehyde dehydrogenase ALDH1a1 and could lead to the accumulation of toxic intermediates of dopamine catabolism. By extrapolating our findings to a potential clinical situation, we suggest that therapeutic downregulation of α-synuclein expression in PD patients is a generally safe option as it should not cause adverse side effects on the functionality of their nigrostriatal system. However, if started in aged patients, this type of therapy might trigger slight functional changes of the nigrostriatal system with potentially unwanted additive effect to already existing pathology.

Genotoxicity of two new carbazole derivatives with antifungal activity.

The class of carbazoles includes compounds with high biological activities and broad spectra of action. PLX01107 and PLX01008 are xenomycins, a new subclass of antimicrobial carbazole derivatives demonstrating strong antifungal activity in vitro. We performed three tests, a bacterial reverse mutation assay (Ames test), in vitro cytokinesis-block micronucleus assay, and chromosome aberration test in mouse bone marrow cells, to investigate the possible genotoxicity of these compounds. Despite their structural similarity, the two compounds had different genotoxicity profiles. PLX01008 showed positive effects in all assays. PLX01107 showed no mutagenicity in the Ames test but demonstrated strong cytogenetic activity in vitro and in vivo. PLX01107 was also tested in the in vivo alkaline comet assay, where a weak but statistically significant increase in DNA damage was seen in liver cells 24h after treatment. Significantly increased levels of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites were found in bone marrow cells of PLX01107-treated mice (FPG-modified comet assay), suggesting induction of oxidative or alkylation damage to DNA.

Investigation of genotoxic and cytotoxic effects of micro- and nanosized titanium dioxide in six organs of mice in vivo.

Titanium dioxide is manufactured worldwide in large quantities for use in a wide range of applications including as food additives, in cosmetics and pigments for coloring ingested and externally applied drugs. Although TiO(2) is chemically inert it can cause negative health effects, such as lung cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. In the present research genotoxicity and carcinogenicity of titanium dioxide were studied in a mouse model. We treated CBAB6F1 mice by oral gavage with titanium dioxide particles (microsized, TDM, 160nm; nanosized, TDN, 33nm) in doses of 40, 200 and 1000mg/kg bw, daily for seven days. Genotoxic effects were analyzed in the cells of brain, liver and bone marrow by means of the Comet assay and in the cells of bone marrow, forestomach, colon and testis with a poly-organ karyological assay (analysis of micronuclei, nuclear protrusions, atypical nuclei, multinucleated cells, mitotic and/or apoptotic index). TDM induced DNA-damage and micronuclei in bone-marrow cells and TDN induced DNA-damage in the cells of bone marrow and liver. TDM and TDN increased the mitotic index in forestomach and colon epithelia, the frequency of spermatids with two and more nuclei, and apoptosis in forestomach (only TDN) and testis. This is one of the first poly-organ studies of TDM- and TDN-induced genotoxicity in vivo in mice. These effects are caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the increasing use of TiO(2) nanoparticles, these findings indicate a potential health hazard associated with exposure to TiO(2) particles.