Adam Holownia

Acetaminophen metabolism and cytotoxicity in PC12 cells transfected with cytochrome P4502E1

Abstract Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen biotransformation by CYP2E1 stimulates this pathway.

Effect of angiotensin IV on the acquisition of the water maze task and ryanodine channel function

In the present study, we investigated the effect of angiotensin IV (Ang IV) on the acquisition of spatial task by rats, expression and function of ryanodine receptors (RyRs) and on Ca(2+) transport in microsomal membranes isolated from rat hippocampus, the brain structure essential for spatial memory.Wistar rats, injected intracerebroventricularly with 1 nmol of Ang IV or saline were subjected to the water maze training using hidden (learning) or visible (nonlearning) escape platform. Rats showed overall good acquisition of the task and mean escape latency decreased from 55 s to less than 10 s during the 5-day training. Learning significantly increased [3H]-ryanodine binding to microsomal RyRs and markedly decreased both receptor affinity constant for the ligand and microsomal Ca(2+) uptake. Ang IV was without effect on the rate of acquisition of the spatial task but increased (by 47%) maximal ryanodine binding in hippocampal microsomes of the trained rats. The peptide, however, did not affect decreased net Ca(2+) uptake in rats subject to learning procedure. Since microsomal Ca(2+)-ATPase activity was similar in all tested groups, the lower net Ca(2+) uptake in the trained rats could be attributed to the elevated expression of RyRs and resulting to increased Ca(2+) release.

The role of calcium in paracetamol (acetaminophen) cytotoxicity in PC12 cells transfected with CYP4502E1

Paracetamol-induced toxicity is mainly due to the accumulation of its CYP450-mediated N-hydroxylation product - N-acetylimidoquinone. We examined cell viability, proliferation rates and intracellular calcium in PC12 cells and in a PC12 cell line transfected with cytochrome P4502E1 exposed to paracetamol. This drug had a concentration-related effect on cell survival and a LD50 which was significantly different between both cell types. A 48% decrease of PC12 cells was found following application of 5 mmol/L paracetamol for 48 h. A total 73% decrease in cell numbers was found in cells metabolizing the drug. Culture protein levels were diminished in a similar manner. Paracetamol increased intracellular calcium (by 662%) only in CYP4502E1-transfected cells. The protective role of EGTA and verapamil modulating calcium homeostasis was more evident in CYP4502E1-transfected cells. These results suggest that biotransformation of paracetamol by CYP2E1 increases its cytotoxicity and that a calcium imbalance may have a key role in the initiation of cell injury.

The effect of ethanol and acetaldehyde on microsomal and mitochondrial membrane fatty acid profiles in cultured rat astroglia.

It has been shown that free radical damage may be involved in ethanol‐induced cytotoxicity in cultured neural cells. Since changes in oxidative metabolism and the resulting lipid peroxidation readily modify biological membranes and alter cell functions we studied the effect of ethanol and its metabolite acetaldehyde on rat astroglial fatty acids profiles in the most common lipid classes of mitochondrial and microsomal membranes, i.e. phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Rat astroglial cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To examine acetaldehyde effects we used a 4‐day co‐culture model consisting of astroglial cells and alcohol dehydrogenase‐transfected Chinese hamster ovary (CHO) cells. Acetaldehyde produced by these cells reached 172 mu M and 265 mu M, respectively, for ethanol concentrations of 10 and 20 m M. Mitochondrial and microsomal membranes were prepared by differential centrifugation, phosphatidylcholine and phosphatidylethanolamine were separated using thin layer chromatography and fatty acid quantitation was performed by GLC. Neither ethanol nor acetaldehyde changed the mitochondrial phosphatidylcholine or phosphatidylethanolamine profiles of total saturated, mono‐unsaturated or polyunsaturated fatty acids. However, some significant alterations in particular fatty acids appeared especially after acetaldehyde but also after the highest ethanol dose. In microsomal phosphatidylcholine monounsaturated fatty acids were significantly increased after both, ethanol and acetaldehyde exposure. Among polyunsaturated fatty acids, arachidonic acid was found to be especially affected by both ethanol and acetaldehyde. Similar decreases were observed in adrenic, docosapentaenoic and docosahexaenoic acids in the groups treated with ethanol. In microsomal phosphatidylethanolamine, ethanol and acetaldehyde decreased monounsaturated and some polyunsaturated fatty acids. These data support the role of peroxidative processes in cultured rat astroglia exposed to ethanol and point to the role of acetaldehyde in this mechanism.