Effects of the pyrrolizidine alkaloid senecionine and the alkenals trans-4-OH-hexenal and trans-2-hexenal on intracellular calcium compartmentation in isolated hepatocytes.

The pyrrolizidine alkaloid senecionine has been shown to produce an increase in cytosolic free Ca2+ concentration in isolated hepatocytes that correlated with an increase in cellular toxicity. The cytotoxicity was greater in the absence of extracellular Ca2+ than in its presence, suggesting that alterations in intracellular Ca2+ distribution, and not an influx of extracellular Ca2+, were responsible for the senecionine-induced hepatotoxicity. The effect of senecionine, as well as the effects of trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and a related alkenal, trans-2-hexenal, on the sequestration of Ca2+ in mitochondrial and extramitochondrial compartments were examined in isolated hepatocytes. Each of the test compounds elicited a decrease in the available extramitochondrial Ca2+ stores that was inhibited by pretreatment with the thiol group reducing agent, dithiothreitol. Senecionine and t-4HH decreased the level of Ca2+ sequestered in the mitochondrial compartment of hepatocytes. The presence of a pyridine nucleotide reducing agent, beta-hydroxybutyrate, inhibited this reduction. These results suggest that both senecionine and t-4HH inhibit the sequestration of Ca2+ in extramitochondrial and mitochondrial compartments possibly by inactivating free sulfhydryl groups and oxidizing pyridine nucleotides respectively.

Lipid peroxidation and cellular damage caused by the pyrrolizidine alkaloid senecionine, the alkenal trans-4-hydroxy-2-hexenal, and related alkenals.

Lipid peroxidation was examined as a possible mechanism for cell injury by trans-4-OH-2-hexenal, the macrocyclic pyrrolizidine alkaloid senecionine and related alkenals in isolated rat hepatocytes. Each compound elicited a positive dose response for peroxidation of cellular lipids as measured by the formation of thiobarbituric acid-reactive products. The addition of the anti-oxidant N,N'-diphenyl-p-phenylenediamine to the hepatocyte suspensions inhibited the production of thiobarbituric acid-reactants. However, the presence of the anti-oxidant had no protective effects on the cell membrane integrity as evidenced by the leakage of lactate dehydrogenase from the cells into the surrounding media. These results suggest that lipid peroxidation which occurs in the presence of senecionine, trans-4-OH-2-hexenal or related alkenals is not entirely responsible for the cellular damage in isolated rat hepatocytes.

Role of cellular calcium homeostasis in toxic liver injury induced by the pyrrolizidine alkaloid senecionine and the alkenal trans-4-OH-2-hexenal.

The pyrrolizidine alkaloid senecionine has been shown to be hepatotoxic, genotoxic, and cytotoxic. However, the biochemical mechanism by which senecionine produces hepatocellular toxicity remains to be elucidated. The role of calcium homeostasis in toxic liver injury was examined in isolated rat hepatocytes treated with senecionine and trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and appropriate cofactors. Hepatocytes treated with senecionine and t-4HH demonstrated greater cytotoxicity (leakage of lactate dehydrogenase) when incubated in the absence of extracellular Ca2+ than in its presence. Both compounds elicited an increase in cytosolic Ca2+ levels of isolated hepatocytes in the presence of extracellular Ca2+. In the following study, senecionine and t-4HH depleted intracellular glutathione levels and induced lipid peroxidation and cytotoxicity in isolated hepatocytes. Pretreatment with the thiol-group reducing agent dithiothreitol prevented depletion of intracellular glutathione and protected hepatocytes against senecionine and t-4HH-induced lipid peroxidation and cytotoxicity. Both compounds also depleted intracellular ATP and NADPH levels. These results suggest that hepatotoxicity induced by senecionine and t-4HH is not dependent on the influx of extracellular Ca2+; however, alterations in intracellular Ca2+, possibly associated with depletion of intracellular glutathione, NADPH, and ATP, may play a critical role.

Genotoxicity and cytotoxicity of selected pyrrolizidine alkaloids, a possible alkenal metabolite of the alkaloids, and related alkenals.

Recently our laboratory isolated trans-4-OH-2-hexenal from the hepatic microsomal metabolism of the macrocyclic pyrrolizidine alkaloid (PA) senecionine and demonstrated in vivo that hepatic necrosis occurred following injection into the hepatic portal vein. To demonstrate similarities in the toxic effects of these compounds, as well as additional macrocyclic PAs and alkenals, genotoxicity and cytotoxicity were examined in primary cultures of rat hepatocytes. A positive cytotoxic response was exhibited by senecionine, retrorsine, seneciphylline, 19-OH-senecionine, trans-4-OH-2-hexenal, trans-4-OH-2-nonenal, and nonenal as measured by the release of LDH. A weaker response was elicited by hexenal. Dosages used of each of these compounds ranged from 30 to 600 nmol/10(6) cells, with each compound exhibiting a linear dose response within this range. All eight compounds exhibited a positive, dose-related genotoxic response as measured by autoradiographic detection of unscheduled DNA synthesis. These results would predict a carcinogenic role for both the PAs and the alkenals. This would suggest similarities in the mechanisms of action of the PAs and alkenals, lending support to the proposed role of trans-4-OH-2-hexenal as an important toxic metabolite of the PAs.

Blood and urine concentrations of cyclizine by nitrogen-phosphorus gas-liquid chromatography.

Gas-liquid chromatography with nitrogen-phosphorus detection was used to analyze blood and urine from a volunteer who ingested 50 mg of cyclizine hydrochloride. A peak blood cyclizine concentration of 69 ng/mL was observed 2 hr after drug administration, and the levels declined thereafter in a biphasic manner, with estimated half-lives of 7 and 24 hr for the early and late phases, respectively. The peak urine cyclizine concentration of 12.5 ng/mL was achieved at 4 hr after administration; only 0.01% of the dose was excreted in the 24 hr urine. Norcyclizine was not observed in blood or urine; however, the detectability of the method for this metabolite is poor relative to the parent drug.