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1,2-unsaturated pyrrolizidine alkaloids (PAs) represent a large group of secondary plant metabolites exhibiting hepatotoxic, genotoxic, and carcinogenic properties upon bioactivation. To examine how the degree of esterification affects the genotoxic profile of PA we investigated cytotoxicity, histone H2AX phosphorylation, DNA strand break induction, cell cycle perturbation, micronuclei formation, and aneugenic effects in different cell models. Analysis of cytotoxicity and phosphorylation of histone H2AX was structure- and concentration-dependent: diester-type PAs (except monocrotaline) showed more pronounced effects than monoester-type PAs. Cell cycle analysis identified that diester-type PAs induced a S-phase arrest and a decrease in the occurrence of cells in the G1-phase. The same structure-dependency was observed by flow-cytometric analysis of PA-induced micronuclei in CYP3A4-overexpressing V79 cells. Analysis of centromeres induced by lasiocarpine in the micronuclei by fluorescence in situ hybridization indicated an aneugenic effect in V79 h3A4 cells. Comet assays revealed no significant induction of DNA strand breaks for all investigated PAs. Overall, diester-type PAs induced more pronounced effects than monoester-type PAs. Furthermore, our results indicate aneugenic effects upon exposure towards lasiocarpine in vitro. These data improve our understanding how structural features of PA influence the genotoxic profile. Especially, the monoester-type PAs seem to induce less severe effects than other PAs. • Pyrrolizidine alkaloids induce structure-dependently cytotoxic & genotoxic effects. • Pyrrolizidine alkaloids induce a cell cycle arrest in S-phase. • Lasiocarpine induced micronuclei show centromeres indicating an aneugenic mechanism. [ABSTRACT FROM AUTHOR]

Recombinant infectious coxsackievirus B3 (CVB3) particles were generated by packaging of modified viral genomes in which the capsid coding P1-region was replaced by an EGFP-luciferase reporter gene. Efficient packaging of the recombinant genome was achieved by a novel method based on cotransfection of a plasmid encoding the subgenomic viral replicon together with two alternative helper plasmids carrying expression cassettes of the CVB3 capsid proteins, and a T7 RNA polymerase expression plasmid. Transcription of a reporter gene and expression of capsid proteins were achieved in a single step, eliminating the need of a helper virus. Recombinant viral stocks were used to infect human embryonal cardiomyocytes (hCMC) and other cell types, and luciferase activity was measured at different timepoints after infection. Neither progeny virus nor wildtype CVB3 was produced upon infection of target cells, facilitating analyses of infected cells without viral spread.The presence of an IRES sequence upstream of the P1 open reading frame in the helper plasmids was indispensable for the generation of recombinant particles, as no packaging was observed using helper plasmids without this feature. Luciferase data obtained by transfection of reporter plasmids with and without upstream 5′-NTR sequences suggests that the CVB3 IRES facilitates translation in T7 RNA polymerase-dependent gene transcription, both in presence and absence of viral replication. [Copyright &y& Elsevier]