In this issue Selectively Toxic Platinum Although we generally think of toxicity in negative terms, selective toxicity is a valuable property. Selective toxicants form the foundation of our modern pesticides, antibiotics, antifungal, antiparasitic, and antitumor agents. Understanding the basis of selective toxicity is key to our ability to design better agents that will only kill the target cell or organism with minimal side effects to the host. Thus, Gao et al. (p 1705) have synthesized a series of isomeric N,N′-bis-salicyl-diaminocyclohexanes (DACHs) and their Pt(II) complexes and evaluated them for their selective toxicity against tumor cells. A primary goal is to develop agents with higher antitumor selectivity than the presently available Pt-based drugs, cisplatin, carboplatin, and oxaliplatin, and to understand the basis of the selectivity.
The three model compounds, 4, 5, and 6, differed in the configuration of the amino groups on the cyclohexyl ring, bearing the (R,R), (S,S,), and (R,S) configurations, respectively. Each compound was cytotoxic against the tumorigenic MCF7 breast cancer cell line, but much less toxic toward nontumorigenic MCF10A breast epithelial cells. All three compounds exhibited higher cytotoxicity and selectivity than cisplatin and oxaliplatin. The enhanced cytotoxicity of compounds 4-6 relative to cisplatin and oxali-
platin correlated with a higher accumulation of Pt in treated cells. Particularly significant was the increased level of DNAbound Pt, for the DACH complexes, consistent with the hypothesis that DNA damage is a major mechanism of cytotoxicity for Ptbased antitumor agents. Gao et al. next investigated the effect of compounds 4-6 on the expression of key genes that modulate the cell cycle and apoptosis. Compound 4, the most cytotoxic, downregulated the expression of cyclin D1, p27, p53, Bcl-xL, and Bcl-2 while increasing the expression of Bax and p21. This pattern of gene expression changes is consistent with cell cycle arrest and the induction of apoptosis. Similar but not identical changes were also observed with compounds 5 and 6, though these compounds were generally less potent. If DNA damage is critical to the cytotoxicity of Ptbased antitumor agents, then the ability of the compounds to access the nuclear compartment is important. Gao et al. synthesized fluorescent-tagged analogues of compounds 4-6 to monitor their uptake and localization in MCF7 cells. They found that the cellular uptake of the labeled analogues correlated with the cytotoxicity of the parent compounds, 4-6. The localization of the fluorescence in the cells suggested uptake by an endocytotic mechanism and confirmed that some of the intracellular compound had accessed the nucleus. The findings suggest that the DACH-derived Pt com-
Published online 10/19/2009 • DOI: 10.1021/tx900308x © 2009 American Chemical Society
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plexes may form the basis for a new class of antitumor agents with higher selectivity than the presently available Pt-based drugs. Further evaluation of these compounds and their mechanism of toxicity will be an interesting topic for future studies. Importance of Methylation It is generally accepted that some of the toxic effects associated with exposure to inorganic arsenic are, in fact, caused by its mono-, di-, and trimethylated metabolites. The enzyme primarily responsible for this metabolism is arsenic (+3 oxidation state) methyltransferase (As3mt). To better understand the role of this enzyme in As distribution and elimination, Drobna et al. (p 1713) have developed As3mt-null mice and evaluated As disposition in these animals.
Drobna et al. administered an oral dose of As as arsenate to wild-type (WT)
and As3mt-null mice and then used high pressure liquid chromatographyinductively coupled plasma mass spectrometry to measure As-containing species in the liver after 2 and 24 h. The results showed that As3mt-null mice retained higher levels of hepatic As than WT mice at both time points. Furthermore, the predominant forms of As in As3mt-null mice were the inorganic and monomethylated species, as compared to WT mice in which substantial dimethylation occurred. The urinary excretion of total As-containing species was similar in WT and As3mt-null mice, but again, the proportion of methylated to inorganic species was much higher in the urine from WT mice. Analysis of radioarsenic contents of various organs 24 h after the administration of [73As] revealed that As3mt-null mice retained higher quantities of the metalloid in liver, kidneys, lungs, heart, and brain than WT mice. Kinetic analysis of total body radioactivity following [73As] administration revealed a biphasic first-
Special Features CRT’s February special issue on Drug Metabolites In Safety Testing continues to generate interest. Now a new review by Walker et al. (page 1653) outlines a systematic approach to the evaluation of metabolite toxicity. In this approach, metabolite identification and assessment occurs at every stage of the drug development process using methods that do not require absolute quantification of metabolites at early stages of the process. The goal is to identify metabolites of concern prior to large scale clinical trials so that patient safety can be assured within the confines of a reasonable investment of resources. Vol. 22,
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In this issue order elimination, with WT and As3mt-null mice clearing 97% and 42-44% of the Asdoseduringthefirstphase, respectively. Together, the data confirm an important role for As3mt in As metabolism. However, the finding of some monomethylated As in As3mt-null mice suggests other mechanisms by which methylation may occur, possibly through the action of bacterial flora in the gut. Nevertheless, it is clear that As3mt-dependent methylation is an important mechanism for the clearance of As from the body, suggesting that this process plays a beneficial role in addition to its contribution to As toxicity. Model Chiral Nerve Agents Organophosphate inhibitors of acetylcholinesterase (AChE) are used as insecticides and as nerve agents for chemical warfare. Efforts to mitigate the effects of nerve agents have focused on developing enzymes that can act as scavengers by binding to and reacting with the compounds in order to prevent their reaction with AChE. This approach is complicated by the fact that the major classes of nerve agents possess a chiral center at the phosphorus atom. Any scavenging enzyme must exhibit the same enantioselectivity for the nerve agent as AChE. However, nerve agents are unavailable for testing, and most model insecticide organophosphates lack the chiral center. To address this problem, Barakat et al. (p 1669) have synthesized a series of enantiomerically enriched nerve 1650
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agent model compounds. This series included compounds bearing the same functional groups as the nerve agents sarin, cyclosarin, and soman; however the fluorine leaving group of the nerve agents was replaced with a thiomethyl or thiocholine group.
Kinetic studies revealed that the Sp enantiomers of all compounds were more potent inhibitors of AChE than the Rp enantiomers. Stereoselectivity was greatest for the cyclohexyl-containing compounds. The thiocholine series was more potent than the thiomethyl series. Parallel studies with butyrylcholinesterase yielded the same general results. Barakat et al. note that thiocholine is a better leaving group than thiomethyl, which partly explains the higher reactivity of the thiocholine series of compounds. Furthermore, the positive charge of the thiocholine moiety helps these compounds to bind in an anionic pocket in the AChE active site that binds the choline moiety of the natural substrate.
Molecular modeling studies showed that the thiomethyl compounds could bind in two orientations, one favoring the thiomethyl and the other favoring the alkoxyl as leaving group. This was consistent with product identification studies that verified both reaction pathways for these compounds.
CHEMICAL RESEARCH IN TOXICOLOGY
(See the accompanying paper by Gilley, et al., p 1680.) In contrast, binding of the thiocholine-containing compounds strongly favors the thiocholine as leaving group. For these compounds, the basis of the stereoselectivity is clear, because this orientation places the bulky alkoxyl group in a confined binding pocket for the Rp enantiomer, whereas the Sp enantiomer places the small, easily accommodated methyl group in this pocket. The data confirm the usefulness of these compounds to serve as models for nerve agents in the future development of scavenger enzymes that can be used to protect against their deadly toxic effects. Pb2+ and Ion Transport Lead is a toxic heavy metal that poses a major public health problem around the world. The nervous system is a primary target of the toxic effects of Pb, possibly resulting from its ability to inhibit the Na+,K+-ATPase. This critical membrane-bound enzyme exchanges intracellular Na+ for extracellular K+ ions against their concentration gradients, using ATP hydrolysis as the energy source. The resulting membrane potential is essential for the action potentials by which neurons transmit electrical impulses. Although Pb-dependent inhibition of the Na+,K+-ATPase is well recognized, the mechanism of the inhibition is not completely understood. Thus, Gramigni et al. (p 1699) conducted a study using membrane fragments containing the ATPase adsorbed on a solid supported membrane. This system al-
lowed them to measure the effects of Pb2+ on electric currents resulting from the addition of Na+ alone (due to the binding of Na+ to the ATPase), Na+ and ATP (due to Na+ transport with ATP hydrolysis), or Na+, K+, and ATP (due to the exchange of both ions with hydrolysis of ATP). Experiments were also performed to measure enzymatic ATP hydrolysis (i.e., ATPase activity) under the same experimental conditions.
The results revealed that Pb2+ inhibited ATPase activity when Na+ and K+ were present in the buffer solution. ATPase activity was also inhibited by Pb2+ in the presence of Na+ only. However, Pb2+ had no direct effect on Na+ binding or transfer except at very high concentrations. Furthermore, the transfer of phosphate from ATP to the enzyme active site, a key step in ATPase catalysis was not influenced by Pb2+.
The data suggest that Pb2+ blocks dephosphorylation of the ATPase active site. K+ binding normally facilitates ATPase dephosphorylation. Perhaps Pb2+ prevents the conformational change that normally occurs with K+ binding. TX900308X
Published online 10/19/2009 •
DOI: 10.1021/tx900308x $40.75 © 2009 American Chemical Society
