Letter

Vsevolod E. Kostrubsky, Ramin Rahbari, Alberto Lodola and Thomas Davies

Departments of Drug Safety Evaluation, Pfizer Global Research and Development, Ann Arbor, Michigan, 48105, and New London, Connecticut, 06320

To the Editor:

In the article by Tirmenstein et al. (2002)Go the authors conclude that the initiating event in troglitazone (TRO) mediated toxicity to HepG2 cells is disruption of mitochondria. We agree with the authors that the relevance of their findings to idiosyncratic hepatotoxicity in TRO-treated patients is uncertain. However, in our view the stringency of their data is not sufficient to conclude that mitochondrial toxicity is an initiating event in TRO-induced cell toxicity.

Our concerns are as follows:

(1) Cell toxicity as measured by LDH release occurred at 50 µM TRO but not at 25 µM TRO. However, there were similar decreases in both mitochondrial potential (as measured by monitoring JC-1) and ATP concentrations at 25 µM TRO and 50 µM TRO. If mitochondrial damage was an initiating event "that preceded cell death" as early as 1 h, then why was there no cell death at 25 µM TRO by 5 h?

(2) Cyclosporin A (CyA) may prevent mitochondrial damage by inhibiting mitochondrial permeability transition (MPT). Cyclosporin A inhibited cytotoxicity and inhibited ATP loss at 50 µM TRO but did not protect against loss of mitochondrial potential at 50 µM TRO. The authors speculate that this was due to CyA-insensitive depolarization. However, CyA has diverse intracellular effects including inhibition of calcineurin-mediated cell death that raises the possibility of nonmitochondrial routes of cell death. It would be interesting to see whether the CyA effect is reproducible in primary liver cells.

(3) It is unclear whether the structures seen after 1 h at 25 µM in the confocal microscopy study are "swollen, round, discrete mitochondria." Their size and morphology are consistent with a nonspecific compartmentalization of dye. In addition, calcein-AM fluorescence was not altered. Calcein-AM should be demonstrated to be in the mitochondria, since it would be expected to redistribute into the mitochondria if there was severe mitochondria membrane damage (Byrne et al., 1999Go).

(4) In contrast to human hepatocytes, HepG2 cells contain low levels of P450s (Feierman et al., 2002Go; Yoshitomi et al., 2001Go; Jover et al., 1998Go). HepG2 cells may also differ in distribution of drug metabolizing enzymes. These differences could result in a unique profile of metabolites in HepG2 cells that could lead to toxicity not relevant to humans.

(5) Mechanisms of cell death may differ in normal and cancer cells. For example, compounds may exert antiapoptotic effects in primary hepatocytes yet proapoptotic effects in proliferating liver cancer cells (Ansorena et al., 2002Go) suggesting coimajor differences in mechanisms of drug-induced cell death in HepG2 cells versus normal hepatocytes.

In conclusion, the data presented by Tirmenstein et al.(2002)Go are inconsistent in terms of mitochondrial effects and, in our view, do not demonstrate that mitochondrial toxicity is the initiating event in idiosyncratic hepatotoxicity seen in TRO-treated human patients.

REFERENCES

Ansorena, E., Garcia-Trevijano, E. R., Mrtinez-Chantar, M. L., Huang, Z., Chen, L., Mato, J. M., Iraburu, M., Lu, S. C., and Avila, M. A. (2002). S-Adenosylmethionine and Methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells. Hepatology 35, 274–279.[ISI][Medline]

Byrne, A. M., Lemasters, J. J., and Nieminen, A. L. (1999). Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes. Hepatology 29, 1523–1531.[ISI][Medline]

Feierman, D. E., Melnikov, Z., and Zhang, J. (2002). The paradoxical effect of acetaminophen on CYP3A4 activity and content in transfected HepG2 cells. Arch. Biochem. Biophys. 398, 109–117.[ISI][Medline]

Jover, R., Bort, R., Gomez-Lechon, M. J., and Castell J. V. (1998). Re-expression of C/EBP{alpha} induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells. FEBS Lett. 431, 227–230.[ISI][Medline]

Tirmenstein, M. A., Hu, C. X., Gales, T. L., Maleeff., B. E., Narayanan, P. K., Kurali, E., Hart, T. K., Thomas, H. C., and Schwartz, L. W. (2002) Effects of troglitazone on HepG2 viability and mitochondrial function. Toxicol. Sci. 69, 131–138.[Abstract/Free Full Text]

Yoshitomi, S., Ikemoto, K., Takahashi, J., Miki, H., Namba, M., and Asahi, S. (2001). Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicity. Toxicol. in Vitro 15, 245–256.[ISI][Medline]





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