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The dark side of a ‘detoxification’ mechanism
News & Comment
TRENDS in Pharmacological Sciences Vol. 22 No.1 January 2001
11
Journal Club
The dark side of a ‘detoxification’ mechanism Drug metabolism is not always a detoxification; sometimes products are generated that are chemically reactive and can damage cells. In the case of acetaminophen [paracetamol (APAP)], although ‘safe’ metabolites are generated at low doses, once enough drug is ingested the reactive metabolite N-acetyl-pbenzoquinoneimine (NAPQI) can be formed. Fortunately, the cell has an abundant nucleophile, reduced glutathione (GSH), which can act as an antioxidant (forming GSSG in the process) or directly scavenge chemicals such as NAPQI, facilitating their excretion as inactivate conjugates. In addition, glutathione S-transferases (GSTs) accelerate GSH conjugation and thereby usually serve to protect the cell. Unfortunately, in APAP overdose this system is overwhelmed, GSH is depleted, crucial targets are apparently damaged by reactive metabolites1 and hepatotoxicity ensues. Although it has been recognized for some time that in certain cases even GS-conjugates can be reactive, Henderson et al.2 have recently proposed another way in which having a GST is not necessarily a good thing. Henderson et al. used mice in which both of the GST Pi family enzymes, GST P1 and GST P2, had been knocked out, and showed that this gene deletion did not aggravate APAP toxicity as had been expected; instead, the GstP1/P2 −/− mice showed little or no signs of hepatoxicity, whereas the
wild-type animals were clearly sensitive to APAP. GSH levels dropped rapidly in both groups but recovered faster in null animals. No significant difference was observed in overall drug–protein adduct formation. xenoprotective enzymes [...] might be involved in modulating cell signalling So, why was the absence of GST Pi enzymes protective? The enhanced recovery of GSH levels suggested that null mice were better able to maintain GSH levels under oxidative challenge. GST Pi enzymes might preferentially make a reversible ipso GS-adduct, and if the ipso adduct reacted with protein thiols (in addition to GSH), particularly those on crucial targets, three effects could ensue: APAP could be regenerated, GSH could be consumed and specific proteins could be inactivated by the formation of mixed disulfides, all of which could be potentiated by the presence of a catalytic GST. Although there was no evidence of GST Pi mediation of GSH conjugation in this report, other studies have suggested a role for rat and human Pi enzymes in the conjugation of NAPQI. GST Pi has also been identified as a target of adduct formation. Henderson et al. propose an alternative theory, namely that GST Pi, acting as a monomer, blocks activation of JUN by inhibiting a JUN N-terminal kinase (JNK).
JUN has been implicated in responses to stress, and APAP has been shown to induce this activity. This highlights a hitherto unexpected role for xenoprotective enzymes: they might be involved in modulating cell signalling. It has been proposed that, under conditions of stress, GST Pi forms dimers and multimers that cannot bind to JNK, thus relieving the inhibition on signalling. It is therefore consistent that a complete absence of GST Pi should potentiate the stress response even further. The challenge now is to determine which, if either, mechanism is responsible for this unexpected effect of the GstP1/P2 −/− knockout animal. A key point will be whether this effect is common to other cases of chemical-induced toxicity. Whatever the outcome, it is clear that even the best detoxification mechanisms can show a dark side under the right conditions. 1 Qui, Y. et al. (1998) Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J. Biol. Chem. 273, 17940–17953 2 Henderson, C.J. et al. (2000) Increased resistance to acetaminophen toxicity in mice lacking glutathione S-transferase Pi. Proc. Natl. Acad. Sci. U. S. A. 97, 12741–12745
Elizabeth M.J. Gillam [email protected]
P2X3 receptors and vanilloids in the micturition reflex pathway The P2X3 receptor, a cation channel that is activated by extracellular ATP, is the predominant ATP receptor subtype found in small-sized dorsal root ganglion (DRG) neurons. Because no potent and selective antagonists of purine nucleotide-gated ion channels (P2X receptors) are currently available, the construction of mice that lack the P2X3 receptor (P2rx3 −/−) offers an invaluable tool to gain information about the role of ATP in sensory mechanisms. ATP and acetylcholine represent the main excitatory neurotransmitters released from postganglionic parasympathetic neurons in the urinary bladder. ATP is also released from
transitional epithelium in response to bladder distension, and can affect the firing of bladder sensory nerves; this prompted the idea of a chemical link between passive mechanical activity, active motor activity and sensory nerves. Immunohistochemical data have shown that P2X3 receptors are found exclusively on bladder nerve fibers, whereas the P2X1 receptor is the most abundant on detrusor smooth muscle. Interestingly, in some DRG neurons, P2X3 receptors colocalize with vanilloid (VR1) capsaicin receptors. Cockayne et al.1* have shown that the urinary bladder of P2rx3 −/− mice is normal upon microscopic analysis (with no
hypertrophy). In normal mice, P2X3 receptors were detected on nerve fibers within the urothelium and bundles in the suburothelial plexus; these receptors were absent in P2rx3 −/− mice, but the sensory nerve network was still intact, as shown by VR1 immunoreactivity. Transvesical cystometries performed in conscious, catheter-implanted animals indicated that P2rx3 −/− mice had an increased bladder capacity compared with their normal counterparts, whereas the amplitude of *This article is also reviewed by Jim Deuchars in the January issue of TINS.
http://tips.trends.com 0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
TRENDS in Pharmacological Sciences Vol. 22 No.1 January 2001
11
Journal Club
The dark side of a ‘detoxification’ mechanism Drug metabolism is not always a detoxification; sometimes products are generated that are chemically reactive and can damage cells. In the case of acetaminophen [paracetamol (APAP)], although ‘safe’ metabolites are generated at low doses, once enough drug is ingested the reactive metabolite N-acetyl-pbenzoquinoneimine (NAPQI) can be formed. Fortunately, the cell has an abundant nucleophile, reduced glutathione (GSH), which can act as an antioxidant (forming GSSG in the process) or directly scavenge chemicals such as NAPQI, facilitating their excretion as inactivate conjugates. In addition, glutathione S-transferases (GSTs) accelerate GSH conjugation and thereby usually serve to protect the cell. Unfortunately, in APAP overdose this system is overwhelmed, GSH is depleted, crucial targets are apparently damaged by reactive metabolites1 and hepatotoxicity ensues. Although it has been recognized for some time that in certain cases even GS-conjugates can be reactive, Henderson et al.2 have recently proposed another way in which having a GST is not necessarily a good thing. Henderson et al. used mice in which both of the GST Pi family enzymes, GST P1 and GST P2, had been knocked out, and showed that this gene deletion did not aggravate APAP toxicity as had been expected; instead, the GstP1/P2 −/− mice showed little or no signs of hepatoxicity, whereas the
wild-type animals were clearly sensitive to APAP. GSH levels dropped rapidly in both groups but recovered faster in null animals. No significant difference was observed in overall drug–protein adduct formation. xenoprotective enzymes [...] might be involved in modulating cell signalling So, why was the absence of GST Pi enzymes protective? The enhanced recovery of GSH levels suggested that null mice were better able to maintain GSH levels under oxidative challenge. GST Pi enzymes might preferentially make a reversible ipso GS-adduct, and if the ipso adduct reacted with protein thiols (in addition to GSH), particularly those on crucial targets, three effects could ensue: APAP could be regenerated, GSH could be consumed and specific proteins could be inactivated by the formation of mixed disulfides, all of which could be potentiated by the presence of a catalytic GST. Although there was no evidence of GST Pi mediation of GSH conjugation in this report, other studies have suggested a role for rat and human Pi enzymes in the conjugation of NAPQI. GST Pi has also been identified as a target of adduct formation. Henderson et al. propose an alternative theory, namely that GST Pi, acting as a monomer, blocks activation of JUN by inhibiting a JUN N-terminal kinase (JNK).
JUN has been implicated in responses to stress, and APAP has been shown to induce this activity. This highlights a hitherto unexpected role for xenoprotective enzymes: they might be involved in modulating cell signalling. It has been proposed that, under conditions of stress, GST Pi forms dimers and multimers that cannot bind to JNK, thus relieving the inhibition on signalling. It is therefore consistent that a complete absence of GST Pi should potentiate the stress response even further. The challenge now is to determine which, if either, mechanism is responsible for this unexpected effect of the GstP1/P2 −/− knockout animal. A key point will be whether this effect is common to other cases of chemical-induced toxicity. Whatever the outcome, it is clear that even the best detoxification mechanisms can show a dark side under the right conditions. 1 Qui, Y. et al. (1998) Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J. Biol. Chem. 273, 17940–17953 2 Henderson, C.J. et al. (2000) Increased resistance to acetaminophen toxicity in mice lacking glutathione S-transferase Pi. Proc. Natl. Acad. Sci. U. S. A. 97, 12741–12745
Elizabeth M.J. Gillam [email protected]
P2X3 receptors and vanilloids in the micturition reflex pathway The P2X3 receptor, a cation channel that is activated by extracellular ATP, is the predominant ATP receptor subtype found in small-sized dorsal root ganglion (DRG) neurons. Because no potent and selective antagonists of purine nucleotide-gated ion channels (P2X receptors) are currently available, the construction of mice that lack the P2X3 receptor (P2rx3 −/−) offers an invaluable tool to gain information about the role of ATP in sensory mechanisms. ATP and acetylcholine represent the main excitatory neurotransmitters released from postganglionic parasympathetic neurons in the urinary bladder. ATP is also released from
transitional epithelium in response to bladder distension, and can affect the firing of bladder sensory nerves; this prompted the idea of a chemical link between passive mechanical activity, active motor activity and sensory nerves. Immunohistochemical data have shown that P2X3 receptors are found exclusively on bladder nerve fibers, whereas the P2X1 receptor is the most abundant on detrusor smooth muscle. Interestingly, in some DRG neurons, P2X3 receptors colocalize with vanilloid (VR1) capsaicin receptors. Cockayne et al.1* have shown that the urinary bladder of P2rx3 −/− mice is normal upon microscopic analysis (with no
hypertrophy). In normal mice, P2X3 receptors were detected on nerve fibers within the urothelium and bundles in the suburothelial plexus; these receptors were absent in P2rx3 −/− mice, but the sensory nerve network was still intact, as shown by VR1 immunoreactivity. Transvesical cystometries performed in conscious, catheter-implanted animals indicated that P2rx3 −/− mice had an increased bladder capacity compared with their normal counterparts, whereas the amplitude of *This article is also reviewed by Jim Deuchars in the January issue of TINS.
http://tips.trends.com 0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.