* National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and Discovery Toxicology, Bristol Myers Squibb Company, Princeton, New Jersey, 08543
Received November 29, 2004; accepted November 29, 2004
The advent of genomic technologies has facilitated major advances in our understanding of the molecular details of normal biology and holds the promise of providing new insights into molecular mechanisms of a variety of toxicities. However, with only a few exceptions, much of the available toxicogenomic data has to date been limited to a description of alterations in gene expression patterns and the development of hypotheses for further evaluation. The article highlighted in this issue (Sawada et al., pp. 282292) represents an important advancement in the application of toxicogenomics in two ways. First, it provides new mechanistic insight into an important issue, namely drug-induced phospholipidosis, and second, the authors have used the validated genomic results to develop an in vitro, rapid screening system to evaluate the potential for compounds to elicit this effect.
Drug-induced phospholipidosis was first identified in 1948 (Nelson and Fitzhugh, 1948), and since that time, about 50 drugs have been shown to produce this phospholipid storage disorder.
Examples of compounds known to elicit this response include the antimalarial chloroquine, the antiestrogen tamoxifen, the antibiotics gentamycin and erythromycin, and several antihistamines. These compounds are cationic amphiphilic drugs (CADs) which are characterized by a primary or substituted nitrogen that is positively charged at physiological pH and a hydrophobic region, usually an aromatic or aliphatic ring. Phospholipidosis is characterized by an intracellular accumulation of phospholipids, primarily in lysosomes in the target organ. Major organs that are affected include liver, kidney, and lung. The condition is difficult to diagnose, relying on the electron microscopic assessment of the appearance of lamellar bodies (Halliwell, 1997; Martin et al., 1989
; Reasor, 1989
; Reasor and Kacew, 2001
). However, although the toxicity can be described, the biochemical mechanisms by which CADs lead to the development of the condition is not fully understood. Furthermore, although the toxicological implications of drug-induced phospholipidosis are unclear at the present time, it is of concern in the development of new drug entities (Reasor and Kacew, 2001
).
From a mechanistic point of view, it is generally recognized that CADs inhibit lysosomal phospholipases, but how they do this is uncertain. The drugs may bind to the phospholipid substrates thereby hindering degradation by the phospholipase enzyme. Alternately, they may bind the phospholipase directly and inhibit the normal function of the enzyme. It is also possible that the drugs increase phospholipid synthesis by an as yet undefined mechanism (Reasor and Kacew, 2001). To date, these mechanisms have been evaluated in a univariate manner, which although informative, has limited major advances in our understanding of this toxicity.
In contrast to the more classical toxicological studies, Sawada et al. (this issue) performed large-scale gene expression analyses in order to understand more completely the pathogenesis of drug-induced phospholipidosis using human hepatoma HepG2 cells. The results established four important pathways that were altered and which contribute to the development of phospholipidosis. Altered gene expression was consistent with lysosomal phospholipase inhibition with increased expression of phospholipid degradation genes such as N-acylsphingosine amidohydrolase 1 and sphingomyelin phosphodiesterase. There was also a role for reduced lysosomal enzyme transport demonstrated by decreased expression of genes such as adaptor-related protein complex 1 sigma 1 which transports lysosomal enzymes between golgi network and the lysosyme. The results also indicated a role for increased phospholipid and cholesterol biosynthesis (stearoyl-CoA desaturase and HMGCoA synthase, respectively). Both of these are triggers for phospholipidosis. Thus, phospholipidosis results from the combination of events involving both increased synthesis and decreased degradation of phospholipids.
Although the genomic data identified important mechanistic events, the authors extended this work to the practical application of developing an in vitro screening test. To this end, they identified a set of 12 marker genes for predicting phospholipidosis. This approach differs from other recent attempts at establishing biomarkers of phospholipidosis in that they used genomics technology in place of analytical chemistry techniques (Mortuza et al., 2003) or metabonomic applications (Nicholls et al., 2000
). These marker genes included functions of phospholipids degradation, cholesterol biosynthesis, fatty acid transport, proteolysis, and endopeptidase inhibition. More importantly, these marker genes proved to provide an accurate analysis of drug-induced phospholipidosis, and the in vitro format required less compound than standard in vivo tests.
The use of genomic technology in toxicology is a burgeoning field, with emphasis on the application of such data for mechanistic studies and biomarker development. However, the utility of such data in regulatory toxicology and risk assessment is equally important (Cunningham et al., 2003; MacGregor, 2003
), and the generation and publication of high-quality, practical application of toxicogenomics is critical to the advancement of the science. The work of Sawada et al. (2005) demonstrates the utility of toxicogenomic approaches for understanding the mechanistic aspects in toxicology and provides a valuable platform for the development of biomarkers of toxicity.
NOTES
1 To whom correspondence should be addressed. Fax: (919) 541-4632. E-mail: cunning1{at}niehs.nih.gov
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