RESUMEN
Phytochemicals like pyrrolizidine alkaloids (PAs) can affect the health of humans and animals. PAs can occur for example in tea, honey or herbs. Some PAs are known to be cytotoxic, genotoxic, and carcinogenic. Upon intake of high amounts, hepatotoxic and pneumotoxic effects were observed in humans. This study aims to elucidate different toxicokinetic parameters like the uptake of PAs and their metabolism with in vitro models. We examined the transport rates of differently structured PAs (monoester, open-chained diester, cyclic diester) over a model of the intestinal barrier. After passing the intestinal barrier, PAs reach the liver, where they are metabolized into partially instable electrophilic metabolites interacting with nucleophilic centers. We investigated this process by the usage of human liver, intestinal, and lung microsomal preparations for incubation with different PAs. These results are completed with the detection of apoptosis as indicator for bioactivation of the PAs. Our results show a structure-dependent passage of PAs over the intestinal barrier. PAs are structure-dependently metabolized by liver microsomes and, to a smaller extent, by lung microsomes. The detection of apoptosis of A549 cells treated with lasiocarpine and monocrotaline following bioactivation by human liver or lung microsomes underlines this result. Conclusively, our results help to shape the picture of PA toxicokinetics which could further improve the knowledge of molecular processes leading to observed effects of PAs in vivo.
Asunto(s)
Alcaloides de Pirrolicidina , Animales , Carcinógenos/farmacología , Humanos , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Alcaloides de Pirrolicidina/química , ToxicocinéticaRESUMEN
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 V79h3A4 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.
Asunto(s)
Histonas , Alcaloides de Pirrolicidina , ADN , Daño del ADN , Hibridación Fluorescente in Situ , Alcaloides de Pirrolicidina/química , Alcaloides de Pirrolicidina/toxicidadRESUMEN
Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.