s2fieldsofstudy: Biology Medicine

text:
Bench-to-bedside review: Molecular pharmacology and clinical use of inert gases in anesthesia and neuroprotection

In the past decade there has been a resurgence of interest in the clinical use of inert gases. In the present paper we review the use of inert gases as anesthetics and neuroprotectants, with particular attention to the clinical use of xenon. We discuss recent advances in understanding the molecular pharmacology of xenon and we highlight specific pharmacological targets that may mediate its actions as an anesthetic and neuroprotectant. We summarize recent in vitro and in vivo studies on the actions of helium and the other inert gases, and discuss their potential to be used as neuroprotective agents.

Introduction
Th e inert or noble gases helium, neon, argon, krypton and xenon exist as monatomic gases with low chemical reactivity. Considerable attention has focused on the use of xenon as a general anesthetic [1][2][3][4] and its potential for use as a neuroprotectant [5][6][7].
A number of recent studies report that helium may have neuroprotectant and/or cardioprotecant properties [8][9][10][11][12][13]. Argon also appears to be neuroprotective in certain in vitro and in vivo models [14,15]. At fi rst sight it might appear unlikely that inert gases would have any biological activity. Nevertheless, evidence for the biological eff ects of inert gases emerged from research into the physiological eff ects of diving. As long ago as the 1930s, nitrogen was shown to be the cause of the narcosis experi enced by divers [16,17]. Th e narcotic eff ects of nitrogen begin to occur at a depth of about 30 meters (a pressure of ~3 atm), and increased with depth, with loss of consciousness occurring at depths of about 100 meters [18,19]. Behnke and Yarbrough showed in 1938 that if helium replaced nitrogen in the breathing mixture, the nitrogen narcosis was avoided [20]. Neon is also devoid of narcotic eff ect [18]. Th e lighter inert gases helium and neon therefore appear both chemically and biologically inactive, at least at tolerable pressures (see below). Argon and krypton, on the other hand, induce narcosis more potently than nitrogen [17,21] -with the pressures resulting in anesthesia being 15.2 atm and 4.5 atm, respectively [22]. Th ese heavier inert gases therefore do have biological activity, at least under hyperbaric conditions. Xenon was predicted to be an anesthetic at atmospheric pressure, based on its relative solubility in fat compared with argon, krypton and nitrogen. An eff ect of xenon in animals was fi rst shown by Lawrence and colleagues in 1946, who reported sedation, ataxia and other behavioral eff ects in mice exposed to between 0.40 and 0.78 atm xenon [21]. Th e anesthetic potency of inert gases follows the Meyer-Overton correlation with solubility in oil or fat (see Figure 1 and Table 1), with xenon being most potent (and most soluble in oil) followed by krypton and argon. Radon is the heaviest of the inert gases and might be predicted to be an anesthetic. Radon is radioactive, how ever, and exposure to radon -even at very low levelsis a health risk [23].
Th e lighter inert gases neon and helium are not anesthetics [24,25], at least up to the highest pressures (~100 atm) that can be tolerated before the confounding eff ects of high-pressure neurological syndrome become pronounced. At these high pressures, the manifestations of high-pressure nervous syndrome include hyper excitability, tremors and convulsions [26,27], which would act to oppose any sedative or anesthetic eff ect. Th e lack of observable anesthetic eff ects of helium and neon are either due to a lack of biological activity or, alternatively, these gases could have some intrinsic anesthetic potency at high pressures that is counteracted by the eff ects of high-pressure nervous syndrome. If we make the assumption that these gases do have some intrinsic potency that would be observable in the absence of the Abstract In the past decade there has been a resurgence of interest in the clinical use of inert gases. In the present paper we review the use of inert gases as anesthetics and neuroprotectants, with particular attention to the clinical use of xenon. We discuss recent advances in understanding the molecular pharmacology of xenon and we highlight specifi c pharmacological targets that may mediate its actions as an anesthetic and neuroprotectant. We summarize recent in vitro and in vivo studies on the actions of helium and the other inert gases, and discuss their potential to be used as neuroprotective agents.
confounding eff ects of high-pressure nervous syndrome, it is possible to calculate a theoretical anesthetic pressure. Based on the Meyer-Overton correlation and using loss of righting refl ex in mice as the anesthetic endpoint, the predicted anesthetic pressures are 156 atm for neon and 189 atm for helium (see Figure 1).

Pharmacology of xenon
Although the general anesthetic properties of xenon have been known since the 1950s, only recently have mole cular targets for xenon been identifi ed that could mediate xenon's biological actions. Th e fi rst target to be identifi ed was the N-methyl-d-aspartate (NMDA) receptor when, in 1998, it was shown that xenon inhibited NMDA-evoked currents in cultured hippocampal neurons by ~60% at a clinically relevant concentration of 80% xenon [28]. Xenon was also found to inhibit NMDA receptors at glutamatergic hippocampal synapses by ~60%, but to have little eff ect on synaptic αamino-3-hydroxy-5-methyl-4-isoxazole pro pi onic acid (AMPA)/kainate receptors [28]. Th e speci fi city of xenon for the NMDA-mediated component of the glutamatergic synaptic response, together with the lack of eff ect at inhibitory γ-amino-butyric acid (GABA)ergic synapses [28,29], imply that xenon acts post synaptically.
Another fi nding consistent with a postsynaptic site of action for xenon is the lack of eff ect of xenon on N-type voltage-gated calcium channels, which are involved in neurotransmitter release at neuronal synapses [30]. Th e molecular mechanism by which xenon inhibits the NMDA receptor has now been elucidated [31]. It has been shown that xenon competes for the binding of the co-agonist glycine at the glycine site on the NMDA receptor ( Figure 2a). Based on protein crystallographic data, the binding of glycine is proposed to result in domain closure of the NMDA receptor leading to channel opening, and competitive inhibitors are suggested to prevent this domain closure [32]. Xenon therefore possibly stabilizes the open conformation of the domains, thus preventing channel opening.
Interestingly, recent crystallographic data on the binding of xenon to the Annexin V protein suggest that xenon may disrupt conformational changes in this protein [33]. Consistent with competitive inhibition at the NMDAreceptor glycine site, xenon inhibits the NMDA receptor more potently at low glycine concentrations than at high glycine concentration (Figure 2b). In addition to competitive inhibition at the glycine site, a Lineweaver-Burk  analy sis (Figure 2b) shows that xenon has an additional noncompetitive component of inhibition [31]. It is possible that xenon's mixed competitive and noncom peti tive inhibition underlies its benefi cial profi le compared with other NMDA receptor antagonists.
It was recently reported that xenon inhibits synaptic AMPA receptors in brain slices from the prefrontal cortex and spinal cord to a similar degree as NMDA recep tors [34] -in contrast to previous studies that found little or no inhibition of AMPA-mediated synaptic responses in hippocampal neurons [28,29]. Th e extent to which AMPA receptors are inhibited by xenon remains to be clarifi ed. If xenon does inhibit AMPA receptors, how ever, this inhibition could contribute to xenon anesthesia and neuroprotection.
Unlike most general anesthetics (for example, iso fl urane, sevofl urane, propofol, etomidate), xenon has little or no eff ect on GABA A receptors. In cultured hippo campal neurons and mouse fi broblast cells stably expres sing α 1 β 1 γ 2L subunits, xenon has no eff ect on currents elicited by exogenous GABA [28]. Similarly, xenon has no eff ect on GABAergic synapses in cultured hippocampal neurons [29]. A study using Xenopus oocytes expressing α 1 β 2 γ 2S subunits, however, reported a small (~15%) poten tiation of GABAevoked currents by xenon [35]. Whether this refl ects diff erences between Xenopus oocytes and mammalian systems or between diff erent GABA A -recep tor subunit combinations is not clear. Nevertheless, xenon's eff ect on GABA A receptors is minimal compared with other anesthetics that typically potentiate GABAergic currents by 100% or more at clinical concentrations [29,[36][37][38][39].
Th e identifi cation of xenon as an inhibitor of the NMDA receptor provided the fi rst putative target for xenon anesthesia and prompted the idea that xenon might be neuroprotective (as glutamate excitotoxicity is involved in pathological conditions such as ischemia and traumatic brain injury [40,41]). Since then a small number of other targets have been identifi ed that may also play a role in mediating xenon's anesthetic and neuroprotective properties.
potassium channels modulate neuronal excitability by providing a background or leak potassium conduc tance. Activation of two-pore domain potassium channels will tend to hyperpolarize the cell membrane and reduce neuronal excitability. Volatile anesthetics such as halothane and iso fl urane also activate TREK-1 [43]. Studies using TREK-1 knockout animals have implicated this channel in general anesthesia with volatile anesthetics, and in neuro protection by the fatty acid linolenate [44]. Whether activation of TREK-1 plays a role in mediating anesthesia and neuroprotection with xenon remains to be determined. Nevertheless, TREK-1 is a plausible target for these actions of xenon.
Recently, xenon has been shown to activate another potassium channel, the plasmalemmal ATP-sensitive potas sium (K ATP ) channel [45]. K ATP channels are inhibited by physiological levels of ATP and act as sensors of metabolic activity. In neurons, K ATP channels are activated under conditions of physiological stress such as hypoxia. Activation of K ATP channels reduces neuronal excitability and is protective against ischemic injury [46]. Clinical concentrations of xenon activate K ATP channels by up to 50% (Figure 2d), and this activation may mediate xenon preconditioning against ischemic injury [45].
Other ion channels that appear to be sensitive to xenon are neuronal nicotinic acetylcholine (nACh) receptors and 5-hydroxytryptamine type 3 (5-HT 3 ) receptors. Neuronal nACh receptors, composed of α 4 β 2 subunits, and homomeric α 7 subunits are inhibited by xenon, where as α 4 β 4 -containing receptors are insensitive to xenon [36,47]. Although nACh receptors are inhibited by a number of anesthetics at clinically relevant concentrations, it is unclear whether this inhibition has any role in mediating general anesthesia. Neuronal nACh receptors have been implicated in neuroprotection (for a review see [48]). However, it is activation of nACh receptors that is neuroprotective. Hence, inhibition of nACh receptors by xenon is unlikely to play any role in xenon neuroprotection. Xenon inhibits human 5-HT 3 receptors expressed in Xenopus oocytes by ~65% at clinical concentrations [49]. Th e clinical signifi cance of this observation, however, is unclear. While 5-HT 3 antagonists, such as ondansetron, are used as antiemetics, xenon appears if anything to cause more postoperative nausea and vomiting compared with propofol [50].

Clinical use of xenon
Xenon was fi rst used as a general anesthetic in the 1950s by Cullen and coworkers in the United States. Th ey reported successful anesthesia in two patients using 80% xenon, 20% oxygen. One patient was an 81-year-old male undergoing orchidectomy, and the other was a 38-yearold female undergoing ligation of the fallopian tubes [51]. Th is was followed by use in a further fi ve patients under going hernioplasty [52]. Loss of consciousness occurred when patients breathed 50% xenon, and a xenon concen tration of 75 to 80% was used for maintenance of anesthesia during the surgery. Following the defi nition of minimum alveolar concentration as the standard anesthetic endpoint by Eger and colleagues [53], the value for xenon was determined. In a study of 28 patients, the minimum alveolar concentration of xenon was found to be 71% [54]. More recent estimates of the xenon minimum alveolar concen tration are in the range 63 to 68% [55,56]. For the next two decades the use of xenon as a general anesthetic remained a curiosity and received little attention.
In the 1990s interest in xenon anesthesia received new impetus as xenon's benefi cial clinical properties were further investigated. Lachmann and coworkers found that xenon anesthesia resulted in greater hemodynamic stability compared with nitrous oxide [57,58]. Th e same studies showed xenon to be a profound analgesic, as evidenced by a greatly reduced need for fentanyl anal gesia during surgery. On average, patients receiving xenon needed only 20% of the dose of fentanyl required when nitrous oxide was used instead of xenon [57]. Similar fi ndings were later reported by Nakata and colleagues [59]. A multi-modal experimental pain study in healthy volunteers reported that the analgesic potency of xenon was 1.5 times that of nitrous oxide [60]. Emergence from xenon anesthesia is rapid, with xenon emergence times being only 50% of the emergence times using nitrous oxide/sevofl urane anesthesia, and the emergence times with xenon are independent of the duration of anesthesia [61,62]. Th ese properties of rapid induction and emergence arise from xenon's very low blood/gas partition coeffi cient of 0.115 [63] and its low solubility in lipids (xenon has an oil/gas partition coeffi cient of 1.9; Table 1) compared with other inhala tional agents. For example, isofl urane has a blood/gas coeffi cient of 1.4 and an oil/gas partition coeffi cient of 97, and for sevofl urane these values are 0.69 and 53, respectively [64].
Xenon's properties of cardiovascular stability, rapid onset and emergence from anesthesia, profound anal gesia and the fact that xenon is not metabolized are some of the characteristics of an ideal anesthetic. Xenon would be a useful replacement for nitrous oxide, with the advantage that xenon -being a natural component of the atmosphere -is not a greenhouse gas. Nitrous oxide, on the contrary, is chemically synthesized and is 230 times more potent as a greenhouse gas than carbon dioxide [65]. Furthermore, there are concerns regarding the possible toxic eff ects of nitrous oxide, particularly in pediatric anesthesia (for reviews see [66,67]).
Th e discovery that xenon is an NMDA-receptor antago nist [28] led to the idea that xenon may be neuroprotective. Th e renewed clinical interest in xenon in the past 10 years is due, in large part, to xenon's potential as a neuroprotectant. In 2003 the fi rst multicenter randomized control trial, involving 224 patients in six centers, compared xenon/oxygen with isofl urane/nitrous oxide anesthesia, and concluded that xenon anesthesia is as safe and eff ective as the isofl urane/nitrous oxide regimen, with the advantage that xenon exhibited more rapid recovery [68]. Another study of 20 patients undergoing coronary artery bypass surgery compared the cardiovascular eff ects of xenon with nitrous oxide when used to supplement fentanyl-midazolam anesthesia. Th is study found that xenon provided better hemodynamic stability and preserved left ventricular function better compared with fentanyl-midazolam alone [3].
Studies in both cardiac and noncardiac patients showed that xenon does not impair cardiovascular function and maintains higher arterial pressure compared with propofol [69][70][71]. A recent multicenter trial of xenon compared with isofl urane found that xenon did not impair left ventricular function while isofl urane signifi cantly decreased global hemodynamic parameters [2].
Th ese clinical data show that xenon is safe and eff ective as an anesthetic, with some advantages compared with conventional anesthesia regimens. Th e high cost of xenon and the need for closed-circuit anesthesia with a specialized anesthesia machine, however, will limit xenon's widespread use unless a signifi cant clinical benefi t (for example, neuroprotection) can be found.

Xenon neuroprotection
Overactivation of glutamate receptors is involved in a number of pathological processes. Excessive entry of calcium, mediated by NMDA receptors, triggers biochemical cascades that ultimately lead to neuronal cell death. Th is neurotoxicity due to overactivation of NMDA receptors was termed excitotoxicity by Olney [72], and is believed to underlie the neuronal injury observed in pathological conditions such as stroke and traumatic brain injury. Th ere has, for some time, been evidence that NMDA-receptor antagonists are neuroprotective in in vitro and in vivo brain injury models [40].
Following the discovery that xenon inhibits NMDA receptors, it was shown that xenon could protect neuronal cell cultures against injury induced by NMDA, glutamate or oxygen-glucose deprivation [6]. Th e same study showed xenon to be neuroprotective in vivo against neuronal injury caused by subcutaneous injection of Nmethyl(d,l)-aspartate in rats. Other NMDA-receptor antagonists such as nitrous oxide, ketamine and dizocilpine (MK801) have intrinsic neurotoxicity, but xenon appears devoid of neurotoxic eff ects [73,74]. Xenon has now been shown to aff ord neuroprotection in a variety of mammalian in vitro and in vivo models, including focal cerebral ischemia (mouse), neonatal asphyxia (mouse), neurocognitive defi cit following cardiopulmonary bypass (rat and pig) and traumatic brain injury (mouse) [5,[75][76][77][78][79][80][81] (Figure 3).
Inhibition of the NMDA receptor by xenon is plausible as a mechanism for xenon neuroprotection. Only very recently, however, has a direct connection between NMDA-receptor antagonism and xenon neuroprotection been demonstrated. Banks and colleagues [7] showed that acute xenon neuroprotection in an in vitro model of hypoxia/ischemia can be reversed by elevating the glycine concentration (Figure 4a), consistent with xenon neuroprotection being mediated by inhibition of the NMDA receptor at its glycine site [31]. Interestingly, xenon appears to act syner gistically with the neuroprotective eff ects of both hypothermia and the volatile anesthetic isofl urane [76,82]. Although a mechanistic explanation for this syner gism remains to be determined, isofl uranein addition to its well-known actions at the GABA A receptor -also competes for glycine at the NMDAreceptor glycine site [31]. Th e binding of volatile general anesthetics to proteins increases at lower temperatures due to favorable enthalpic interactions, and this increase in binding correlates with the increase in general anesthetic potency observed at lower temperatures [83][84][85]. Whether xenon exhibits similar temperature dependence in its interactions with the targets mediating its anesthetic and neuroprotective eff ects remains to be elucidated.
In addition to its action as an acute neuroprotectant (when applied during or after the insult), xenon is neuroprotective in preconditioning paradigms. Precondition ing refers to the situation where a neuroprotectant is present before the insult, but not during or after the insult. Exposure to xenon for 2 hours, prior to hypoxia/ ischemia 24 hours later, was shown to result in reduction of injury in cultured neurons and in vivo in neonatal rats [45,86]. Inhibition of the NMDA receptor might be thought to be less likely to play any role in xenon preconditioning, as pathological glutamate release occurs only during and after the insult. Since NMDA receptors are not overstimulated before the insult, how their inhibition by xenon could mediate xenon preconditioning is not as clear as in the case of acute xenon neuroprotection. Nevertheless, NMDA receptors are known to couple to many intra cellular signaling pathways, so it remains possible that xenon inhibition of normal NMDA-receptor functioning before the insult could trigger some longterm eff ect that might mediate preconditioning.
Whether the NMDA receptor plays a role in xenon preconditioning remains to be determined. A recent study, however, has identifi ed the ATP-sensitive potassium K ATP channel as being involved in xenon preconditioning. Bantel and colleagues showed that xenon preconditioning against hypoxia/ischemia is abolished by the plasmalemmal K ATP channel blocker tolbutamide (see Figure 4b) [45], implying a role for the activation of the K ATP channel. Th e mechanism by which transient activation of the K ATP channel by xenon results in neuroprotection 24 hours later is not known. Th ere is some evidence to suggest that xenon preconditioning results in an increase in phosphorylated cAMP response element binding protein and the pro-survival proteins B-cell lymphoma 2 and brain-derived neurotrophic factor [86], although a causal link with K ATP channels has not been established.
Th e clinical trials discussed previously have looked at the safety and effi cacy of xenon as an anesthetic. Very few trials, however, have as yet directly addressed xenon neuro protection. Clinical trials are underway, or planned, looking specifi cally at xenon as a neuroprotectant in cardiopulmonary bypass (a procedure associated with postoperative cognitive defi cit), neonatal asphyxia and neurological defi cit following cardiac arrest and resuscitation. To date, however, none of these trials have been completed.
A phase 1 trial in patients undergoing coronary artery graft on cardiopulmonary bypass that showed xenon can be safely delivered to these patients has been completed [1]. Two trials have examined postoperative cognitive defi cit (POCD) in elderly patients undergoing noncardiac elective surgery, comparing xenon anesthesia with propofol anesthesia [87,88]. Neither study found a decreased incidence of POCD in the xenon group compared with the propofol group. Another study looking at POCD in elderly patients undergoing elective surgery found no advantage of xenon compared with desfl urane anesthesia [89].
Th e lack of effi cacy in these trials may, partly, be explained by the low numbers of patients resulting in underpowered studies. Only one of the studies involved  [78], (b) Homi and colleagues [5], (c) Ma and colleagues [75], and (d) Coburn and colleagues [77].
Dickinson and Franks Critical Care 2010, 14:229 http://ccforum.com/content/14/4/229 more than 100 patients [87], and the other two used fewer than 40 patients each. Another confounding factor is that, although POCD is a recognized phenomenon, particularly in the older person, it is not straightforward to quantify POCD. Th e diff erent studies used diff erent assessment criteria and diff erent times after surgery when assessments were performed. Larger trials will be required to defi nitively determine whether xenon reduces POCD in elderly patients.
POCD following cardiopulmonary bypass (CPB) is thought to result in part from particulate and gaseous cerebral emboli subsequent to CPB. Concerns have been raised about the potential eff ects of xenon on gasembolism growth as xenon may increase the size of preexisting gas emboli, but estimates as to the extent of this eff ect vary widely in the literature. A theoretical study predicted rapid and infi nite expansion of 50 nl air bubbles in the presence of 70% xenon [90]. An experimental study, however, found 50% xenon to have only a relatively modest eff ect [91]. Both this study and other experimental studies have compared xenon with nitrous oxide, and show that xenon causes much less expansion of gas bubbles than does nitrous oxide [91][92][93].
Studies in animal models of CPB have reported diff ering results regarding the eff ects of xenon on gas emboli. Grocott and colleagues reported a modest (17%) increase in the size of large (~400 nl) air bubbles artifi cially introduced into a bypass circuit in a rat model in the presence of 70% xenon [93]. Another study using a rat CPB model combined with artifi cially introduced air bubbles of 300 nl reported that exposure to 56% xenon resulted in increased infarct volume and neurological defi cit compared with nitrogen [94]. A later study by the same group, however, concluded that xenon did not aff ect neurological or histological outcome [95]. Th e reasons for these discrepancies are not clear.
It should be noted that the artifi cial introduction of a small number of relatively large air bubbles into the CPB circuit does not accurately model the clinical scenario, where it is more likely that bubbles will be small in size but may be numerous. Th e only human trial that has directly measured embolic load in CPB patients during xenon treatment found that xenon (20 to 50%) caused no increase in embolic load [1]. Nevertheless, the issue of whether xenon may increase embolic load should be borne in mind (and monitored) in future clinical trials.
Aside from its potential to reduce POCD, xenon could be argued to be more likely to show a benefi t in situations where the potential damage in the absence of any neuroprotection is more severe. In this regard it will be interesting to see whether clinical trials of xenon in neonatal asphyxia show xenon to be neuroprotective, as has been demon strated in in vivo models of neonatal asphyxia [76,96]. Acute xenon neuroprotection against hypoxia/ischemia involves the N-methyl-Daspartate-receptor glycine site. Acute xenon protection is reversed by adding glycine. Applying 50% atm xenon after hypoxia/ischemia in the absence of added glycine (black bars) gives robust protection (32 ± 6% of control injury). However, the protective eff ect of 50% atm xenon is abolished in the presence of 100 μM glycine. Addition of the inhibitory glycine receptor antagonist strychnine (100 nM) had no eff ect on control oxygen-glucose deprivation (OGD) with or without glycine, xenon neuroprotection without glycine, or the reversal of xenon neuroprotection by glycine. The error bars are standard errors from an average of 44 slices at each condition. Data have been normalized to the control OGD with no added glycine. *Value signifi cantly diff erent (P <0.05) from control OGD. n.s., not signifi cant. Figure adapted from Banks and colleagues [7]. (b) Xenon preconditioning against hypoxia/ischemia involves the plasmalemmal ATP-sensitive potassium (K ATP ) channel. Exposure of cultured neurons to 75% xenon for 2 hours protects cells against hypoxia/ischemia 24 hours later (white bar). This eff ect is abolished by the plasmalemmal K ATP blocker tolbutamide (Tb) (0.1 mM) but not by the mitochondrial K ATP channel blocker 5-hydroxy-decanoic acid (5-HD) (0.5 mM). *P <0.05, **P <0.01. Figure adapted from Bantel and colleagues [45].

Use of helium and other inert gases as potential neuroprotectants
Th e evidence for the neuroprotective properties of xenon has prompted interest in investigating whether other inert gases have similar potential as neuroprotectants. Helium is the lightest of the inert gases, is not an anesthetic, is much more abundant and is signifi cantly cheaper to produce than xenon. Mixtures of helium and oxygen (heliox) are used in diving to avoid the eff ects of nitrogen narcosis. Medical use of helium/oxygen has been advocated in patients with respiratory illness. Th e fi rst use of helium/oxygen in acute asthma patients was in 1934 [97], with the study reporting an alleviation of dyspnea. Recent systematic reviews, however, have concluded that the current evidence does not support use of helium/oxygen in acute asthma or chronic obstructive pulmonary disease [98,99], and helium has not been widely used to treat respiratory illness.
To date there have been relatively few studies investigating the potential of helium as a neuroprotectant, and these have been limited to in vitro and in vivo models. In an in vitro organotypic hippocampal brain slice model of traumatic brain injury, mild hyperbaric helium (0.5 or 1 atm) was found to be neuroprotective [77]. Th is study found that the outcome was signifi cantly worse if nitrogen replaced helium. Th e authors concluded that the eff ect of helium was the result of a benefi cial eff ect of pressure per se combined with an attenuation of the deleterious eff ects of nitrogen [77]. Interestingly, an in vitro model of hypoxic/ischemic injury using the same organotypic brain slice preparation found no eff ect of 0.5 atm helium [7]. Th is may refl ect the fact that diff erent mechanisms of injury are activated in these diff erent injury paradigms. Another in vitro study using cultured neurons reported that normobaric helium (75%) was actually detrimental to neuronal survival after hypoxia/ ischemia [15]. An in vivo study in rats subjected to focal ischemia, however, reported that 75% helium reduced the infarct volume and improved functional neurological outcome 24 hours after injury [11]. Th e reasons for the diff ering fi ndings with helium in these studies are not entirely clear.
Nevertheless, it is interesting to note that these variable eff ects with helium contrast with the eff ects observed with xenon, which appears to be neuroprotective in all of these models. While a number of pharmacological targets have been identifi ed for xenon, no targets have been identifi ed for helium.
Helium is considered to be inert and lacking in an intrinsic pharmacological eff ect; helium is therefore often used as a pressurizing gas in studies of the biological eff ects of pressure per se [100,101]. Compared with xenon, which has neuroprotective eff ects at concentrations similar to those causing anesthesia, it seems implausible that the non anesthetic helium would have any pharmacological eff ect at or near atmospheric pressure. Even if we assume, as predicted by the Meyer-Overton correlation, that helium might be anesthetic at ~200 atm (Figure 1), if helium was neuroprotective at 1 atm it would be acting at 1/200th of its anesthetic concentration. Eff ects at such low relative concentrations have not been observed for other anes thetic neuroprotectants. Even in the case of xenon, which is neuroprotectant at subanesthetic concentrations as low as ~20% [76], the ratio of neuroprotectant to anes thetic concentration is only ~1/3. Helium therefore seems unlikely to be acting via a pharmacological mecha nism.
An interesting recent study by David and colleagues, however, has identifi ed a probable physical mechanism that may underlie the reported neuroprotective eff ects of helium [12]. Th is study in rats found that, at room temperature, 75% helium resulted in signifi cantly reduced brain infarct size and improved functional neurological outcome when helium treatment took place following middle cerebral artery occlusion. Th e authors discovered, however, that breathing helium gas below body temperature (for example, 25°C) caused hypothermia in the rats (Figure 5a). Helium was neuroprotective when the inspired temperature was 25°C, but the neuroprotective eff ect was abolished when the temperature of the inspired helium was increased to 33°C (abolishing the hypo thermia) (Figure 5b). Th e authors conclude that the neuroprotective eff ects of helium are due to hypothermia.
Neuroprotection via cooling is well established in model systems and is used clinically (for reviews see [102,103]). Th e reason that helium causes hypothermia is due to its high thermal conductivity compared with air. Th e thermal conductivity of helium is 0.1499 W/m/Kalmost six times greater than nitrogen, which has a thermal conductivity of 0.0260 W/m/K (Table 1). Breathing helium at a temperature lower than body temperature will hence cause a reduction in core temperature. Th is phenomenon is recognized in divers breathing heliox mixtures who require heated diving suits and heated gas delivery equipment in order to avoid hypothermia. Xenon, on the contrary, has a thermal conductivity fi ve times lower than nitrogen (see Table 1), and therefore would not result in cooling via this mechanism. In common with other anesthetics, however, xenon exhibits an anesthesiainduced hypothermia. Th e neuroprotection observed with helium is probably therefore due to helium-induced hypothermia rather than to any pharmacological eff ect of helium. Th e cooling eff ect of helium could also occur in in vitro systems lacking adequate gas-tempera ture control, and this may explain the variable eff ects observed in diff erent studies using helium.
Th e other inert gases -neon, argon and krypton -have received very little attention as potential neuroprotec tants. Argon and krypton are anesthetics under hyper baric conditions, at 15 atm and 4.5 atm, respectively, and might be expected to be neuroprotective at these pressures. It is conceivable that argon and krypton could be neuroprotective at atmospheric pressure -by analogy with xenon, which exhibits neuroprotective properties even at ~1/3 of its anesthetic potency. Neon, on the contrary, is not an anesthetic -but based on its oil solubility, neon might be predicted to be an anesthetic at ~160 atm. By the same argument as above for helium, neon is unlikely to have a pharmacological neuro protective eff ect at atmos pheric pressures. Neon's thermal conductivity is twice that of nitrogen, hence neon breath ing might induce hypothermia. Any eff ect, how ever, is likely to be much less than that caused by helium. Argon does appear to be neuroprotective in certain model systems. In an in vivo study, normobaric argon (25 to 77%) increased survival rates of rats exposed to varying degrees of hypoxia [104]. An in vitro study using cochlear organotypic cultures from rats found that argon (74 to 95%) was protective against hypoxic injury and injury induced by the anticancer drug cisplatin or the antibiotic gentamycin [14]. Another in vitro study using mouse cortical cell cultures found that 75% argon protected against hypoxic/ischemic injury but that the same concen trations of krypton or neon had no eff ect [15]. A recent in vitro study has shown that normobaric argon protects mouse hippocampal organotypic cultures against both ischemic and traumatic injury [105]. Argon there fore does indeed appear to be neuroprotective at normo baric pressures. Th is eff ect is most probably mediated by a pharma co logical mechanism. Th e thermal conductivity of argon is less than that of nitrogen -hence argon will not cause hypothermia via this physical mecha nism, but may cause anesthesia-induced hypothermia at elevated pressures. Th e reason for the lack of neuroprotective eff ect of krypton is unclear. To date, however, there has only been a single in vitro study on krypton.
Whether krypton has a neuroprotective eff ect in other injury paradigms merits further investigation. No molecu lar targets have as yet been identifi ed that could mediate anesthesia or neuroprotection by argon or krypton. Molecular modeling, however, suggests that the inert gases with anesthetic properties (argon, krypton and xenon) and nitrogen all make similar types of interactions with a model protein cavity [106]. Th e binding energies of the inert gases can only arise from favorable enthalpic (ΔH) contributions due to London Dispersion forces (also known as van der Waals interactions) and/or charge-induced dipole interactions. Both of these enthalpy terms are proportional to the polarizability (α) of the gas (Table 1). Relative to a particular standard state, the energy of these favorable enthalpic (ΔH) terms must be suffi cient to overcome the unfavorable entropy term associated with binding. Th e anesthetic inert gases (argon, krypton and xenon) can be distinguished from the nonanesthetic helium and neon by their greater polariza bility [106] (Table 1), which results in larger favorable enthalpic interactions. Xenon, for example, has a value of α of 4.04 Å 3 , which is 19 times greater than that of helium (0.21 Å 3 ) and 10 times that of neon (0.39 Å 3 ). Argon and krypton have α values of 1.64 Å 3 and 2.48 Å 3 , respectively, which are eight times and 12 times greater than the value for helium. Th erefore it is plausible that Figure 5. Helium causes hypothermia in rats, which mediates its neuroprotective eff ect. (a) Breathing 75% helium at temperatures lower than 37°C results in hypothermia. (b) Breathing 75% helium at 25°C following injury protects the cortex against focal ischemic injury (light grey bar). The protective eff ect of helium is abolished if the gas is warmed to 35°C (dark grey bars). The striatum is resistant to both injury and the protective eff ects of hypothermia (shown on the right). *P <0.05. Figures adapted from David and colleagues [12].
argon and krypton interact with the same targets as xenon, even if somewhat more weakly. Th at anesthesia and neuroprotection by the inert gases share similar mechanisms is, therefore, an interesting possibility.

Conclusions
Th e present review summarizes studies on the pharmacology and clinical uses of the inert gases as anesthetics and neuroprotectants. Xenon is the only inert gas that is an anesthetic at atmospheric pressure. A relatively small number of pharmacological targets for xenon have been identifi ed that may play a role in xenon anesthesia and neuroprotection; the NMDA receptor, the two-pore domain potassium channel TREK-1 and the K ATP channel. Xenon has been shown to be an eff ective neuroprotectant in in vitro and in vivo injury models, and the results of clinical trials to assess xenon's eff ectiveness as a neuroprotectant in patients are eagerly awaited. Th e mecha nisms involved in xenon neuroprotection are begin ning to be understood. Th ere is new evidence that inhibition of the NMDA receptor by xenon mediates acute xenon neuroprotection, and that the K ATP channel is involved in xenon preconditioning.
Helium has been shown to be neuroprotective in vivo, but this eff ect is mediated by helium-induced hypo thermia rather than by a pharmacological eff ect. Even if helium is devoid of pharmacological action, the cooling eff ect resulting from helium's high thermal conductivity could be exploited clinically. Furthermore, as xenon and hypothermia appear to act synergistically in experimental models, it is possible that the two neuroprotective strategies of xenon and hypothermia could be applied simultaneously using a helium/xenon mixture combined with an appropriate controlled gas-cooling apparatus.
Argon and krypton are anesthetic at elevated pressures, but few studies have investigated neuroprotection by argon and krypton. However, argon appears to be neuroprotective at atmospheric pressure in certain model systems. Further studies are needed to determine whether argon and krypton have potential as neuroprotectants.


extfieldsofstudy: Biology Medicine

text:
Hepatic progenitors for liver disease: current position.

Liver regeneration restores the original functionality of hepatocytes and cholangiocytes in response to injury. It is regulated on several levels, with different cellular populations contributing to this process, eg, hepatocytes, liver precursor cells, intrahepatic stem cells. In response to injury, mature hepatocytes have the capability to proliferate and give rise to new hepatocytes and cholangiocytes. Meanwhile, liver precursor cells (oval cells) have become the most recognized bipotential precursor cells in the damaged liver. They rapidly proliferate, change their cellular composition, and differentiate into hepatocytes and cholangiocytes to compensate for the cellular loss and maintain liver homeostasis. There is a growing body of evidence that oval cells originate from the intrahepatic stem cell(s), which in turn give(s) rise to epithelial, including oval cells, and/or other hepatic cells of nonepithelial origin. Since there is a close relationship between the liver and hematopoiesis, bone marrow derived cells can also contribute to liver regeneration by the fusion of myeloid cells with damaged hepatocytes, or differentiation of mesenchymal stem cells into hepatocyte-like cells. The current review discusses the contribution of different cells to liver regeneration and their characteristics.

It is believed that the ancient myth of Prometheus describes the capability of the liver to restore its mass. Punished by Zeus for the theft of fire, Prometheus, a Titan, was chained for eternity to a rock in the Caucasus; each day an eagle would eat his liver and each night the liver would regenerate only for the eagle to return the following day.
Liver regulates different functions during growth, development and adulthood. It serves as the first site of embryonic hematopoiesis, but turns into a vital detoxifying system in the postnatal period. In addition, liver serves as a major storage of glycogen and vitamin A, and remains the only organ in the adult organism capable of self renewal. 1 Liver regeneration is a rapid and tightly orchestrated process that efficiently and precisely restores the original cellular mass and functionality of hepatocytes and cholangiocytes. It is regulated on several levels, with different cellular populations contributing to liver regeneration. First, mature hepatocytes (in contrast to mature hematopoietic cells) possess the ability to proliferate and repopulate the damaged liver ( Figure 1). Some studies suggest that hepatocytes can give rise not only to new hepatocytes, but can also differentiate into cholangiocytes, suggesting that hepatocytes are bipotential cells, which possess stem cell-like features. Second, liver precursor cells (oval cells) differ from other cells in the liver by their ability to extensively proliferate, migrate in a specific zone (lobule) and differentiate into hepatocytes and cholangiocytes. The concept of "liver precursor cells" has not been widely accepted until the last decade, most likely due to the established and recognized regenerative potential of hepatocytes. A population of small portal cells with ovoid nuclei and high nuclear/cytoplasmic ratio, named oval cells, was first identified in 1978 by Shinozuka and colleagues 2,3 Since then, oval cells have become the most recognized precursor cells in the liver. The term "oval cells" is widely used to characterize a heterogeneous population of bipotential transient amplifying cells originating in the Canal of Hering, and activated in the liver as a result of injury or insult. [4][5][6] The number of quiescent (or dormant) oval cells is very low under physiological conditions, but in response to an abruptly changing microenvironment, oval cells rapidly proliferate, change their morphology, surface markers and cellular composition, and differentiate to compensate for the cellular loss to maintain liver homeostasis. 7 Third, since oval cells represent a mixed precursor population, they most likely differentiate from the "hypothetical true" intrahepatic stem cell(s). 5,8,9 Here we call the intrahepatic stem cell hypothetical because it has not been well defined, but the growing evidence suggests its existence. Hypothetical intrahepatic stem cell(s) can, therefore, give rise to oval cells, causing their heterogeneity and/or other hepatic cells of non-epithelial origin. 9 Fourth, since there is a close relationship between the liver and hematopoiesis, the contribution of bone marrow (BM)  Figure 1 Cells contributing to liver regeneration. Notes: Different cellular types regulate liver regeneration on several levels. Contribution of mature hepatocytes to regeneration of hepatic parenchymal cells is shown in black. Liver precursor cells with bipotential differentiation capacity are shown in red. Proposed pathways of liver regeneration, which require further clarification, are shown in grey. Abbreviations: HSC, hepatic stem cells; MSC, mesenchymal stem cells.
Current position on hepatic progenitors Dovepress submit your manuscript | www.dovepress.com Dovepress derived cells to liver regeneration has to be considered. Early studies proposed that hematopoetic cells can contribute to the replenishment of hepatic stem cells and oval cells. 10,11 However, under most circumstances, reconstitution of the liver with BM cells results from fusion of damaged hepatocytes with myeloid cells. 12 Furthermore, mesenchymal stem cells derived from the bone marrow or adipose tissues have a capacity to improve liver function, perhaps by giving rise to hepatocyte-like cells. 13,14 Thus, we cannot exclude that circulating (hematopoietic and/or mesenchymal) stem cells cannot ameliorate the intrahepatic stem cells in response to severe injury.

Hepatocytes
In normal adult liver, mature hepatocytes exhibit quiescent phenotype, stay in the G0 phase of the cell cycle, and show minimal turnover. However, upon hepatocyte loss (such as shock liver, liver infection or surgical resection), these mature hepatocytes undergo cell division, while maintaining their metabolic function. [15][16][17][18] The most widely used model to study hepatocyte transplantation, proliferation and reconstitution potential is the fumaryl-aceto-acetate hydrolase (FAH) knockout mice, in which the metabolic disorder causes extensive, continuous liver injury. In this murine model for the human disease, hereditary tyrosinemia type 1, donor hepatocytes expressing the FAH protein have a strong selective growth advantage over the recipient hepatocytes. 12,19 Hepatocyte repopulation in wild type mice or FAH -/mice is very rare. However, high efficiency of hepatocyte repopulation in FAH -/mice is achieved only through massive selection pressure, caused by withdrawal of the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) from drinking water, which results in severe loss of hepatocytes. Therefore the normal lobular structure and liver functions of FAH -/mice can be totally restored by transplantation of wild type hepatocytes. 18,19 This model has demonstrated that normal adult hepatocytes can be serially transplanted through seven generations of mice, and that single hepatocytes can be clonally amplified like stem cells, and serially passaged to repopulate the FAH -/mouse liver. 20 As liver reconstituting cells, mature hepatocytes have the capacity to reconstitute almost 70% of livers in FAH -/mice. 20 Successful transplantation of hepatocytes in mice encourage generation of "humanized" mouse models of liver disease. These models serve as a useful tool to study the physiology of human hepatocytes in mammalian organisms (versus in vitro), and further advance drug development for liver therapy. Several genetically manipulated mice have been successfully used for this purpose. Thus, human hepatocytes, obtained from patients after liver transplantation or by liver biopsies, were transferred into immunodeficient transgenics Alb-uPA(tg(+/-)) that express uroplasminogen activator (uPA) under the transcriptional control of an albumin promoter. 21,22 Another mouse model, Rag2 -/-/Il2rg -/mice (immunodeficient for recombinant activation gene-2 and the common γ-chain of the interleukin receptor mice), which are devoid of T and B cells, have been reported to be excellent recipients of human hematopoietic xenografts. 23 High efficiency of liver repopulation with human hepatocytes can be achieved when Rag2 -/-/Il2rg -/are crossed with Alb-uPA(tg(+/-)) mice. 24 Finally, generation of mice triple mutant for Fah, Rag2 and Il2rg can be efficiently repopulated with human hepatocytes. 25 The stem like properties of differentiating hepatocytes have encouraged development of protocols for hepatocyte transplantation in patients, a procedure which is an appealing alternative to auxiliary orthotropic liver transplantation. Hepatocyte transplantation has been used in attempts to treat genetic diseases, including familial hypercholesterolemia, Crigler-Najjar syndrome type I, glycogen storage disease type 1a, urea cycle defects and congenital deficiency of coagulation factor VII. 26,27 So far, hepatocyte transplantation in humans has yielded only early but not sustained success rates. Problems such as appropriate number of donor hepatocytes for transplantation, rejection of transferred hepatocytes, localization of the engrafted cells, and hepatocyte activity are currently unresolved and require further investigation. Moreover, human adult liver cells are often not available in sufficient numbers for successful repopulation of the donor liver. Human hepatocytes are difficult to maintain in culture. They do not multiply long term in vitro and also do not tolerate cryopreservation well. Alternative resources for cell transplantation could be bipotential liver precursor cells, mesenchymal stem cells, embryonic stem cells (ESC), or inducible pluripotential cells (iPS), expanded and differentiated in vitro into hepatocyte-like cells.

Liver precursor cells (oval cells)
Extensive or chronic liver damage, or prolonged intoxication and viral infection results in hepatocyte senescence, when mature hepatocyte proliferation is exhausted and suppressed. 28,29 In this case, liver regeneration is carried out by a heterogeneous population of progenitor cells, located within (or immediately adjacent to) the canal of Hering, which become activated and proliferate to replace the impaired hepatic parenchyma. 4 These cells were first described by E Faber in 1956 as "small oval cells with scant lightly basophilic cytoplasm and pale blue-staining Dovepress nuclei" with a high nuclear/cytoplasmic ratio and an ovoid nucleus, and were called "oval cells". 6 Oval cells represent a bipotential precursor population, which can simultaneously co-express epithelial markers typical for cholangiocytes (CK-7, CK-19, and OV-6) and hepatocytes (alpha-fetoprotein and albumin), suggesting the close relationship of oval cells to differentiated liver parenchymal cells. 30,31 Experiments tracing the transfer of 3 H in thymidine-labeled oval cells confirm that these cells behave like bipotential progenitor cells and are able to differentiate into hepatocytes and cholangiocytes. [30][31][32][33][34] Once activated and proliferating, it is proposed oval cells give rise to duct-like structures that arise from the periportal regions and spread into the liver acinus; several days after activation these ductlike structures change appearance, and transform into clusters of small basophilic hepatocytes and mature bile ducts.
Several researchers have isolated and immortalized hepatic progenitors. Generation of these bipotential cell lines have helped in studying the molecular mechanisms that regulate and drive activation and differentiation of progenitor cells. In 1998 Spagnoli and colleagues 35 isolated and established the MMH (met murine hepatocyte) palmate cell line; immortalized bipotential liver precursor cells derived from explants of embryonic liver of transgenic mice expressing a constitutively active truncated human Met receptor (cyto-Met) under control of the human α1-antitrypsin transcription unit. 36 Under appropriate culture conditions, Palmate cells are able to differentiate into hepatocytes and cholangiocytes, and are widely used to dissect the molecular pathways of hepatocyte formation. 37 To date, many inbred mouse strains have been used to isolate and manipulate bipotential liver cells 38,39 that possess progenitor properties in vitro and are in vivo and able to differentiate into hepatocytes and colonize diseased livers in mice. 35,38,40,41 Due to significant differences in the anatomical structure of murine and human canals of Hering, oval cells are named liver precursor cells (LPCs) 9,42 in human livers. LPCs proliferate in chronic liver diseases, such as alcoholic disease, steatohepatitis and viral hepatitis. However, LPCs are difficult to track and isolate because of the lack of a known definitive marker. Only recently, Schmelzer and colleagues 43 have been able to isolate LPCs based on their expression of two surface markers, the epithelial cell adhesion molecule (EPCAM) and neural cell adhesion molecule (NCAM). LPCs have the ability to repopulate the liver in animal models.
According to our current understanding of liver regeneration, oval cells or LPCs represent a transient population of amplifying cells derived from normally quiescent true stem cells, which may reside in the biliary tree. 8 In this context, the existence of a mesendodermal stem cell, with broader progenitor properties, capable of differentiation into hepatic parenchymal and non-parenchymal cells, has been hypothesized. 7,44 Purification of hepatic progenitor cells is often based on cell sorting, using putative oval cell specific surface markers. However, identification of oval cells is difficult because the surface and intracellular markers expressed by human and rat oval cells significantly differ from those expressed by mouse oval cells. Thus, rat oval cells express OV6, bile duct epithelium markers (CK-19) and hepatocyte markers (albumin, α-fetoprotein [AFP]) and resemble hepatoblasts. 2 The detailed characterization of oval cells in the rat has been done by Yovchev and colleagues 7 (Figure 2). Rat oval cells express markers described mostly for hepatopoietic stem cells, such as c-kit, Sca-1, CD34 and Thy-1.1. [45][46][47] While this fact would suggest that oval cells or true liver stem cells are replenished from BM progenitor cells, expression of Thy-1 by oval cells remains controversial. [48][49][50] However, a recent study has demonstrated that Thy-1 + cells are located in rats in the "oval cell niche" and represent a subpopulation of activated mesenchymalepithelial cells that are distinct from resident stellate cells, myofibroblasts, or oval cells. 51 Surprisingly, rat and mouse oval cells exhibit remarkable heterogeneity in expression of specific markers. 52 In the search for specific precursor markers, it has been suggested that murine oval cells upregulate CK-19, AFP, albumin, Thy-1, CD34, delta-like protein (Dlk), EPCAM, TROP2, or the rat oval cell marker OV6. 47,[53][54][55][56][57] There is strong evidence independently demonstrated by several groups that mouse oval cells express CD133 and Sca-1. [58][59][60][61][62] However, in contrast to rat oval cells, mouse oval cells do not always express these markers, and until recently could be identified by expression of only one intracellular marker A6. 47,57 Hence, new antibodies against liver precursors may provide new markers of mouse oval cells (MIC1-1C3; OC2-1D11; OC2-2F3; OC2-1C6; OC2-2A6; OC2-6E10). Proliferating cells recognized by the new antibodies were located in ductal versus periductal areas, confirming the heterogenic nature of murine oval cell population 63 and (Conigliaro and colleagues, unpublished observations) (Figure 2).
Many oval cell-specific markers are expressed intracellularly and cannot be used for isolation of viable cells. Moreover, expression of many reported markers, especially in rats and humans, is not restricted to the hepatic precursor cells, and many proposed markers are also upregulated by other cell types. Although oval cells can be isolated using a combination of several surface proteins, their ability to exhibit precursor properties is further tested in vitro and/or in vivo. Since there Current position on hepatic progenitors Dovepress submit your manuscript | www.dovepress.com Dovepress is no agreement in the literature as to which combination of markers defines oval cells, an epithelial colony-forming assay has been developed to assess the precursor properties of isolated cells. By culturing highly enriched precursors with one cell in each well of 96-well plates, the nature of each cell is analyzed. Isolated multipotent precursors have to undergo in vitro expansion prior to differentiation analysis or being reintroduced back into the mouse (via adoptive transfer into the spleen). In vitro expanded precursors are tested for their ability to form colonies. For example, precursors (CD133 + CK19 + CK7 + AFP + CD34 + and CD45 -Ter119c-kit -Thy-1 -), isolated from adult livers of DDC-treated mice or untreated mice, exhibit distinct features of the multipotent population by forming two types of colonies in vitro, large mixed-lineage and small unilineage colonies. 61 Hence, cells from DDC-treated mice form both mixed-and unilineage colonies, which can give rise to hepatocytes and cholangiocytes when transferred into FAH -/mice, while the cells from untreated mice formed only unilineage colonies. In addition, clonal expansion of single cells from the large mixed-lineage colonies has demonstrated that only 1% of the cells possessed true precursor properties. 61

Dovepress
Oval cells represent a heterogeneous population. Three different scenarios can explain the heterogeneity of oval cells and their place in the hierarchy of liver stem cells: 1) Oval cells are a pool of hepatic progenitors undergoing different stages of differentiation. 2) Originating from the liver stem cells, oval cells themselves can follow different pathways of differentiation and give rise to distinct classes of oval cells with highly specialized functions, whose bi-potential activity is carried out by the cholangiocyte or hepatocyte progenitors. 3) Alternatively, oval cells are heterogeneous hepatic precursor cells that may originate from common hepatobiliary stem cells during differentiation upon the needs of hepatic tissue. 9 Liver stem cells Several reports have described the isolation of pluripotent cells from human fetal (human fetal liver multipotent precursor cell, [HFLMPC]) 64 and adult livers. 65 HFLMPCs are classified as mesenchymal-epithelial transitional cells, which can be maintained in long-term culture in an undifferentiated state, but when properly treated differentiate into hepatocytes and cholangiocytes as well as into cells of several mesenchymal lineages (fat, cartilage, bone and endothelial cells). Human liver stem cells (HLSCs) are proposed to be a mesenchymal liver population with a partial commitment to hepatic cells. When cultured in the appropriate differentiation media, HLSCs can be induced to differentiate into mature hepatocytes, osteoblasts, or endothelial cells, showing a multilineage potentiality. Adult liver precursor cells appear in vivo in response to several types of pathological liver injury, especially when hepatocyte replication is impaired. These cells are histologically identified by the expression of cholangiocyte markers and proliferate in the portal area of the hepatic lobule. HLSCs may play an important role in liver regeneration by providing the self-renewing ability of bipotent primitive hepatic cells. 61 The success rate of using the stem cells for restoring tissues depends on the ability to expand these cells in vitro and, in many cases, to direct their differentiation towards a specific cell lineage. [66][67][68][69] Although in vitro expansion, growth and differentiation of isolated fetal stem cells does not necessarily drive their differentiation in vivo, it provides a unique system to study stem cell regulation, and to optimize conditions to direct endogenous regenerative potential towards a lineagespecific differentiation. In this regard, RLSCs (resident liver stem cells), a non-tumorigenic stable stem cell line obtained from fetal and neonatal murine livers, represent a useful tool to study stem cell biology. 70 In their undifferentiated state, RLSCs maintain a stem cell transcriptional profile and ability for self-renewal. Undifferentiated RLSCs co-express mesenchymal (eg, calponin 1), endodermal (eg, FoxA1 and aFP) and neuroectodermal (eg, nestin) stem cell markers together with several "stem" related genes (chromatin remodeling genes) crucial for the activation of stem cell genetic program. RLSCs differentiate into hepatocytes and cholangiocytes in vivo and in vitro and, when cultured under appropriate conditions, can give rise to mesenchymal and neuro-ectodermal cell lineages, such as osteoblasts/osteocytes, chondrocytes, astrocytes and neuronal cells. The ability to spontaneously differentiate into hepatocytes and the lack of albumin expression in an undifferentiated state, places RLSCs at the pre-hepatoblast/liver precursor cell hierarchy of liver stem cells. RLSCs were used 71 to study liver zonation, demonstrating for the first time a direct convergence of the canonical Wnt signaling on the HNF4α driven transcription in RLSC derived periportal hepatocytes.

BM contribution to liver regeneration
Significant contribution of the BM to hepatocyte regeneration was originally established in the FAH -/mice, which develop liver failure in response to withdrawal of NTBC from their drinking water. Transplantation of total BM or myeloid cells into these mice results in fusion with the recipient hepatocytes, their clonal expansion, and reconstitution of normal liver mass. Despite this extraordinary ability of myeloid cells to rescue the phenotype of FAH -/hepatocytes, such phenomenon was not (or very rarely) observed in response to other types of liver injury (CCl 4 , bile duct ligation [BDL]), suggesting that hematopoietic cells have a limited contribution to hepatocyte population under physiological conditions or in response to injury. 72,73 Meanwhile, there is some evidence that hematopoietic stem cells can contribute to replenishment of liver precursors or stem cells, since transplantation of highly purified hematopoietic stem cells resulted in generation of BM-derived ductular cells and hepatocytes. 10,74,75 One can speculate that BM cells can replace oval cells or liver stem cells. In a recent study, Cho and colleagues have investigated the ability of purified hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and mononuclear cells to engraft and contribute to liver regeneration in response to injury. Of these populations, the MSCs transplanted group showed the highest engraftment rate and exhibited the utmost potential to differentiate into hepatocytes when co-cultured with injured liver cells. 76 MSCs have been isolated from several sources, BM, cord blood, amniotic fluid, placenta and adipose tissue, and Current position on hepatic progenitors Dovepress submit your manuscript | www.dovepress.com Dovepress therefore, become a prime candidate for cell therapy. Several studies have demonstrated that MSCs isolated and expanded in vitro can be induced to differentiate into hepatocyte-like cells. 77,78 However, only a low level of liver repopulation with the MSCs, derived from umbilical cord or adipose tissues, has been reported. In addition, differentiation of MSCs into hepatocytes requires both in vitro expansion and treatment with hepatogenic factor prior to transplantation. Several attempts have been made to treat liver pathologies with undifferentiated MSCs. Thus, human umbilical cord blood-derived cells transplanted into non-obese diabeticsevere combined immunodeficient (NOD-SCID) mice generated clusters of donor-derived hepatocytes in response to CCl 4 . 79 Although transplantation of MSCs often results in attenuation or improvement of liver disease, current studies have not provided a definitive proof for MSC differentiation into functional hepatocytes in vivo. 14,77,[79][80][81] Even more controversial is the role of MSCs in fibrosis. In response to fibrogenic liver injury (CCl 4 , BDL), both hematopoietic and MSCs have shown limited contribution to hepatocyte replenishment, but in turn, actively differentiated into fibrogenic cells. 72,73,82 Moreover, Russo and colleagues have reported that MSCs directly differentiate into myofibroblasts in the damaged liver and facilitate fibrogenic processes. 83 Therefore, it remains unclear if infusion of MSC fulfills the function or increases the risk from the transplantation therapy.

Conclusion
This review has summarized the potential contribution of intrahepatic and BM cells to liver regeneration. There are at least three sources of intrahepatic cells that are capable of differentiating into hepatocytes and cholangiocytes. In response to injury, hepatocytes themselves undergo activation, proliferate and differentiate into parenchymal cells. This process is the best documented and most understood, and currently provides the most therapeutic potential. Meanwhile, oval cells, heterogeneous liver precursor cells that most likely arise from the biliary tree, may contribute to newly formed hepatocytes and cholangiocytes. However, very little is known about the biology of oval cells, especially the conditions suitable to manipulate oval cell differentiation and expansion in vitro prior to reintroduction in vivo. Even less is known about "true" liver stem cells and their regenerative potential. Future studies are likely to further characterize intrahepatic stem cells, and new findings are needed to improve cell transplantation therapy. submit your manuscript | www.dovepress.com


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Chloramphenicol Causes Mitochondrial Stress, Decreases ATP Biosynthesis, Induces Matrix Metalloproteinase-13 Expression, and Solid-Tumor Cell Invasion

Overuse and abuse of antibiotics can increase the risk of cancer. Chloramphenicol can inhibit both bacterial and mitochondrial protein synthesis, causing mitochondrial stress and decreased ATP biosynthesis. Chloramphenicol can accelerate cancer progression; however, the underlying mechanisms of chloramphenicol in carcinogenesis and cancer progression are still unclear. We found that chloramphenicol can induce matrix metalloproteinase (MMP)-13 expression and increase MMP-13 protein in conditioned medium, resulting in an increase in cancer cell invasion. Chloramphenicol also activated c-Jun N-terminal kinases (JNK) and phosphatidylinositol 3-kinase (PI-3K)/Akt signaling, leading to c-Jun protein phosphorylation. The activated c-Jun protein has been proven to activate binding to the MMP-13 promoter and also upregulate the amount of MMP-13. Both the SP 600125 (JNK inhibitor) and LY 294002 (PI-3K/Akt inhibitor) can inhibit chloramphenicol-induced c-Jun phosphorylation, MMP-13 expression, and cell invasion. Overexpression of the dominant-negative JNK and PI-3K p85 subunit also negate chloramphenicol-induced responses. Other antibiotics that cause mitochondrial stress and a decrease in ATP biosynthesis also induce MMP-13 expression. These findings suggest that chloramphenicol-induced PI-3K/Akt, JNK phosphorylation, and activator protein 1 activation might function as a novel mitochondrial stress signal that result in an increase of MMP-13 expression and MMP-13-associated cancer cell invasion. The findings of this study confirms that chloramphenicol, and other 70S ribosomal inhibitors, should be administered with caution, especially during cancer therapy.

Chloramphenicol, extracted from Streptomyces venezuelae, is an antibiotic with a small molecular size and highly lipid soluble. Chloramphenicol is indicated for the treatment of wound healing and ocular infections. Chloramphenicol reaches therapeutic concentrations in the cerebrospinal fluid and is used for the treatment of meningitis in the third-world countries.
Also, it is used as the last-line defense for multiple drug-resistant organisms, such as vancomycin-resistant Enterococcus (Hammett-Stabler and Johns, 1998). Aside from its pharmaceutical application, chloramphenicol has become a major contaminant in farmed seafood (especially shrimps, crab, and fish). Due to the fact that some farmed seafood contain excessive chloramphenicol residue, new concerns about its toxicity have been raised. Adverse effects caused by overdose and overuse of chloramphenicol include aplastic anemia, gray baby syndrome, and leukemogenesis (Hammett-Stabler and Johns, 1998;Holt et al., 1993;Robin et al., 1981). In a mouse T-cell model, chloramphenicol induced abnormal cell differentiation and inhibited apoptosis, leading to the development of leukemialike syndrome (Yuan and Shi, 2008). Based on epidemiologic studies, chloramphenicol did not contribute to carcinogenesis when applied topically. However, its causal association with leukemia, especially through systemic use, was probably not adequately tested in these studies (Smith et al., 2000). From a thorough review by the International Agency for Research on Cancer, chloramphenicol is listed as a probable carcinogen to humans (Group 2A). The underlying mechanism of chloramphenicol in human solid-tumor progression is still unclear.
Chloramphenicol reversibly binds to the 50S subunit of the 70S ribosome, inhibiting peptidyl transferase in both prokaryotic organisms and mitochondria (Balbi, 2004). In histopathological studies, chloramphenicol-stressed mitochondria showed morphological and functional deterioration (Duewelhenke et al., 2007). Mitochondrial damage is an important contributor to human aging and aging-related diseases. Thus, we assumed that chloramphenicol-treated somatic cells may show senescencerelated phenotypes. In 2005, we found that chloramphenicol caused senescence-associated morphological changes and senescence biomarker SA-bGal activation in somatic cancer cells (Li et al., 2005). Besides morphological alteration, senescent cells often exhibit cell cycle blockage, decreased sensitivity to growth factors and apoptosis stimulators, and changes in their gene expression pattern. A study using complementary DNA (cDNA) array showed that a variety of genes involved in cell cycle regulation, cytoskeleton, stress response, and metabolism are altered during senescence (Kang et al., 2003).
Upregulation of matrix metalloproteinases (MMPs) during senescence may contribute to matrix degradation and senescent phenotype appearance (Kang et al., 2003). MMPs are a group of zinc-dependent endopeptidases. It has been demonstrated that MMPs participate in several physiological (including embryonic development and wound healing) and pathological events (rheumatoid arthritis and tumor metastasis) (Kuzuya and Iguchi, 2003;Leeman et al., 2002). At least 23 different MMPs have been identified in humans. MMPs have been classified into gelatinases, collagenases, stromelysins, and membranetype MMPs. The MMP-13 (also called collagenase-3) can hydrolyze collagen types I, II, III, IV, X, and XIV; gelatin; aggrecan; tenascin C; and fibronectin (Knäuper et al., 1997a;Leeman et al., 2002). Because of its proteolytic potency and wide substrate specificity, the expression of MMP-13 is restricted to specific tissues, including human fibroblasts, keratinocytes, and solid-tumor cells (Knäuper et al., 1997a). High levels of MMP-13 often correlate with malignant tumor invasion, suggesting an important role for MMP-13 in tumor progression (Knäuper et al., 1997a;Leeman et al., 2002).
The rationale of the study is to verify chloramphenicol-induced senescence-like responses, especially the mitochondria-to-nucleus stress signals and MMP expression in chloramphenicol-treated H1299 cells. We found that chloramphenicol treatment activates PI-3 kinase/Akt/c-Jun N-terminal kinases (JNK) signaling and increases nuclear c-Jun activity, which contribute to MMP-13 expression and H1299 cell invasion.

MATERIALS AND METHODS
Reagents, cell culture, and treatments. All chemicals used were of analytical grade and, unless otherwise indicated, were purchased from Sigma (St Louis, MO). Chloramphenicol was dissolved in ethanol, prepared freshly, and protected from light. The solvent content was < 5 ll/ml in all experiments. The H1299 (non-small-cell lung cancer), HepG2 (hepatocarcinoma), A549 (lung adenocarcinoma), and HaCaT cell (immortalized keratinocyte) were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, and cultured in a humidified atmosphere of 5% CO 2 at 37°C.
Transient transfection of dominant-negative-JNK and DN p85. H1299 cells were seeded in complete medium in six-well plates for 24 h before transfection. Cells were transfected with 2-lg plasmid DNA containing dominant-negative (DN) JNK (a gift from Dr H.-F. Yang-Yen, Institute of Molecular Biology, Academia Sinica, Taiwan) or DN p85 (d-p85; a gift from Dr W. M. Fu, Institute of Pharmacology, National Taiwan University, Taiwan) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in serum-free medium, according to the manufacturer's protocol. The medium was replaced with complete medium 6 h later. Cells were then incubated for another 24 h and were then exposed to chloramphenicol.
Cellular ATP content measurement. Total cellular ATP levels were determined by ATP-bioluminescent assay. Cell lysate was prepared as describe in the manufacturer's protocol (BioVision, Mountain View, CA). Each reaction was performed by mixing 5-10 ll lysates in 90 ll reaction buffer in a 96-well plate. Finally, luciferase and its substrates were added and luminescence intensity was counted immediately, using a luminometer (TopCount; Packard, Ramsey, MN) calibrated with appropriate ATP standards.
RNA isolation, reverse transcription-PCR, and real-time quantitative PCR. Total RNA was isolated by the acid guanidium thiocyanatephenol-chloroform extraction as described previously (Li et al., 2005). To analyze MMP messenger RNA (mRNA) expression, reverse transcription (RT)-PCR were performed by evaluating MMP mRNA content and that of a housekeeping gene, b-actin, as an internal control. In brief, the first strand cDNA was synthesized from 3 lg total RNA at 42°C for 60 min. Specific MMP cDNAs were amplified as follows: denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s and a final 10 min extension at 72°C. The PCR products were separated by 2% agarose gel electrophoresis, with ethidium bromide staining. The PCR primer sets for MMPs and b-actin are listed in Supplementary table 1.
The real-time quantitative PCR (qPCR) and quantitative measurements were performed with SYBR-Green PCR Mix essentially as described by the manufacturer (Bio-Rad, Hercules, CA). For MMP-13, the RT-PCR primer set was purchased from SABiosciences (Frederick, MD). Normalized expression levels were then calculated using the starting levels of b-actin in each sample to normalize for differences in total RNA content in individual samples.
Nuclear protein extraction. Nuclear extracts were prepared using nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). According to the manufacturer's protocol, cell monolayer on 100-mm dishes were scraped and resuspended in cytoplasmic extraction reagent by repetitive pipetting. The cytosolic fraction was separated by centrifugation (15,000 3 g, 5 min). The nuclear protein was extracted with nuclear extraction reagent by intermittently vortexing over 40 min.
In vitro invasion assay. The in vitro invasion assay was carried out in transwell plates with 8-lm pores. Each transwell was precoated with 25-30 ll diluted Matrigel (BD Biosciences, Belford, MA) (1:3 dilution with serum-free DMEM) as described previously (Yagel et al., 1989). H1299 (2.5 3 10 4 cells per well) or A549 (5 3 10 4 cells per well) was seeded on top of the Matrigel layer and incubated for 12-48 h. Noninvading cells and the Matrigel layer were removed using a cotton bud and the migrated cells, attached to the lower membrane, were fixed by immersion in 4% formaldehyde for 3 min. Finally, the invading cells were counterstained with 0.05% crystal violet and counted.
Lung colonization assay. Experimental in vivo metastatic potential of the H1299 (with or without chloramphenicol treatment) was measured by the lung CHLORAMPHENICOL INDUCES MMP-13 EXPRESSION colonization assay (Kim et al., 2009). Cells used for injection were grown to subconfluence, treated with chloramphenicol for 24 h, trypsinized, washed, and single-cell suspensions were prepared in serum-free DMEM. The cell suspensions (5 3 10 4 single cells/0.2 ml DMEM) were injected into the tail vein of 4-to 6-week-old male mice. C57BL/6J mice were obtained from National Taiwan University Animal Center and housed aseptically in the animal facilities. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Twenty days later, the mice were anesthetized with isoflurane and sacrificed. The lungs were fixed with Bouin's solution, and metastatic colonies on the lung surface were counted macroscopically.
Gelatin zymography. The enzymatic activity of MMP-13 was assayed by gelatin zymography (Yoon et al., 2002). Conditioned medium was mixed with sample buffer without reducing agent. The sample was loaded into a gelatin (1 mg/ml)-containing SDS-polyacrylamide gel electrophoresis. After electrophoresis, the gel was washed twice in washing buffer (50mM Tris-HCl, pH 7.4; 150mM NaCl; and 2.5% Triton X-100) for 30 min and then incubated in incubation buffer (50mM Tris-HCl, pH 7.4; 150mM NaCl, 0.02% NaN 3 , 10mM CaCl 2 , and 1lM ZnCl 2 ) at 37°C. After 12 h of incubation, the gel was stained with 0.1% Coomassie brilliant R-250 in 50% methanol and 10% acetic acid for 30 min and destained with staining solution without Coomassie brilliant R-250. The clear zone of gelatin digestion indicated the presence of MMP protein.
Chromatin immunoprecipitation. The assay was performed using the chromatin immunoprecipitation (ChIP) kit (Upstate Biotech, Lake Placid, NY). Living cells were treated with formaldehyde to generate protein-DNA crosslinks. Then, sheer chromatin (with an average DNA fragment size to~500 base pairs) was prepared by repeated sonication. The extract was divided to INPUT and ChIP portions. Anti-phospho-cJun IgG (Catalog sc-822; Santa Cruz) were added to the ChIP portions at 1:100 dilution for immunoprecipitation. The nonspecific IgG was used as a negative control. The immunocomplexes were pulled down by protein A/Sepharose and washed out of the nonspecific binding. The cross-links in the immunoprecipitated chromatin were reversed by heating overnight with proteinase K at 65°C. The DNA was purified by phenol/ chloroform extraction and ethanol precipitation. The purified ChIP-captured DNA of samples and of input DNA samples were amplified by the PCR method as described before. The following oligonucleotides: 5#-CACCCACAT-CAGGAACCCCCCATC-3#, 5#-GAGGAAGACCTCCAGTTTGCA GAG-3# (MMP-13) and 5#-GATATCGCCGCGCTCGTCGTCGAC-3#, 5#-ACCATCAC GCCCTGGTGCCTGGGG-3# (b-actin) were used for PCR detection.
Statistical analysis. All data were expressed as mean ± SD from at least three independent experiments. Statistical analysis was carried out using Student's t-test or one-way ANOVA, and values of p < 0.05 were considered statistically significant.

Chloramphenicol Caused Mitochondrial Stress, ATP Restriction, and Increased Cancer Cell Invasion
Chloramphenicol testing dosages (10-100 lg/ml) used in these studies were without cytotoxicity in MTT assay (Supplementary fig. 1). The clinical dosage of chloramphenicol normally used against infectious diseases is 10-30 lg/ml in serum, which corresponds to a dose of 50-100 mg/kg/day (Balbi, 2004). Chloramphenicol inhibited mitochondrial protein translation (Fig. 1A), caused mitochondrial stress, and decreased ATP biosynthesis (Fig. 1B) in H1299 cells in a dose-dependent manner. A transwell assay showed that the ATP-restricted cell condition could promote cancer cell invasion (Fig. 1C).

Chloramphenicol Induced MMP-13 mRNA Expression in Cancer Cells and Facilitated Cancer Cell Invasion
The mRNA levels of MMPs were analyzed by RT-PCR in chloramphenicol-treated H1299 cells. Cellular MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) mRNA levels Mitochondria inhibitor rotenone was used as positive control. Chloramphenicol treatment decreased ATP biosynthesis in H1299 cell. The ATP restriction condition resembled the hypoxia. (C) To evaluate the invasion capacity of chloramphenicol-treated H1299 cell, the Matrigel-coated 8-lm transwells were used as described in the ''Materials and Methods'' section. The data showed that chloramphenicol-induced ATP-restricted H1299 cell exhibited stronger invasion activity than control. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference with the control.
Both MMP-3 and MMP-13 mRNA expression levels remained unaffected in immortalized normal cells (including HaCaT keratinocytes, CCD-966SK fibroblast, IMR-90, retinal pigmented epithelial cell [ARPE]) and primary cultures (human umbilical vein endothelial cell, human kidney epithelial cell, mouse lung cell, rat cardiocyte, and rat vascular smooth muscle cell) (Supplementary fig. 2). Our findings suggest that the chloramphenicol-induced MMP-13 expression only existed in solid-tumor cells but not in leukemia suspensions and normal cells.

CHLORAMPHENICOL INDUCES MMP-13 EXPRESSION
We also found that the conditioned medium of chloramphenicoltreated H1299 cells could enhance the invasive ability of A549 cells, which have previously been demonstrated to have weak invasive ability (Mooradian et al., 1992). This increase could also be inhibited by CL-82198 (Fig. 3A). During the entire process, the Western blots and gelatin zymography also showed strong proteolytic activity of the MMP-13 protein in conditioned medium with chloramphenicol-treated H1299 cells (Fig. 3B). Next, we investigated the effect of chloramphenicol-induced MMP-13 expression on cancer cell metastasis in vivo. In comparison with H1299, treatment with 100 lg/ml chloramphenicol for 24 h led to a remarkable increase in metastatic colony formation on lung surface. The MMP-13 inhibitor, CL-82198, inhibited metastatic activity of chloramphenicol-treated cells (Fig. 3C). The in vivo data supported that chloramphenicol-induced MMP-13 expression is strongly associated with metastatic capacity of solid-tumor cells.

The Effects of MAPKs and PI-3 Kinase Inhibitors on Chloramphenicol-Induced MMP-13 Expression
To assess the involvement of MAPK and phosphatidylinositol 3-kinase (PI-3K)/Akt signaling in chloramphenicolinduced MMP-13 mRNA expression, cells pretreated with MAPKs and PI-3K inhibitors were cotreated with chloramphenicol and assayed by RT-PCR. Results showed that both SP 600125 and LY 294002 suppressed chloramphenicol-mediated MMP-13 mRNA expression. However, the induction of MMP-13 by chloramphenicol treatment remained unaffected by PD 98059 or SB 203580 (Fig. 4B). The SP 600125 and LY 294002 FIG. 3. The MMP-13 protein in chloramphenicol-treated H1299 condition medium facilitated A549 cell invasion. (A) About 5 3 10 4 A549 cells were seeded into transwells and incubated with condition medium of chloramphenicoltreated H1299 cell for 24 h. The data showed that secreted MMP-13 in condition medium could enhance A549 cells invasion. The increase in invasive A549 cell counts could be prevented by MMP-13 inhibitor, CL-82198 (20lM), but not by MMP-3 inhibitor, NNGH (20lM). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference with the control. # p < 0.05, ## p < 0.01, and ### p < 0.001 indicate a statistical difference with chloramphenicol-treated condition medium control. (B) Chloramphenicol-treated H1299 condition medium was collected, and the protein was precipitated by 10% trichloroacetic acid. MMP-13 protein in condition medium was detected by Western blotting. For gelatin zymography, the condition medium was concentrated. The gelatin enzymatic digestion was performed as described in the ''Materials and Methods'' section. Both MMP-13 protein level and activity were increased in chloramphenicol-treated H1299 condition medium in a time-dependent manner. (C) The in vivo metastatic activity was evaluated by lung colonization assay. The H1299 or chloramphenicoltreated H1299 cells were iv injected into living C57BL/6J mice. Twenty days later, the colonies formation on lung surface was counted. No colony was found in mice received DMEM injection. Treatment with 100 lg/ml chloramphenicol for 24 h led to a remarkable increase of metastatic colonies formation on lung surface compared to H1299 cell control. MMP-13 inhibitor, CL-82198, treatment inhibited metastatic activity to a resting state. ***p < 0.001 indicate a statistical difference with the H1299 control. ## p < 0.01 indicate a statistical difference with chloramphenicoltreated H1299 group. 144 also inhibited MMP-13 activity by gelatin zymography (Fig. 4C) and chloramphenicol-induced cell invasion (Fig. 4D). Chloramphenicol-induced JNK phosphorylation could also be inhibited by LY 294002, whereas SP 600125 did not abolish Akt activation (Fig. 5A). The data suggest that PI-3K/Akt/JNK signaling is essential for chloramphenicolassociated MMP-13 induction and cancer cell invasion.

PI-3 Kinase/Akt/JNK Signaling Required for
Chloramphenicol-Induced c-Jun Phosphorylation and MMP-13 Expression Coincubation with SP 600125 or LY 294002 inhibited chloramphenicol-induced c-Jun phosphorylation in the nucleus. The proliferating cell nuclear antigen (PCNA) protein was used as an internal control in the nuclear extracts (Fig. 5A).
Plasmids encoding DN JNK or the p85 subunit of PI-3K (DN p85) were transfected into H1299 cells. The HA-tag protein confirmed the successful expression of the DN JNK and p85 proteins. In these successfully transfected cells, chloramphenicol-induced JNK activation and c-Jun phosphorylation were reduced, and there was no MMP-13 activity in gelatin digestion (Fig. 5B).

Chloramphenicol Increased Activator Protein 1 DNA-Binding Activity and Induced MMP-13 Expression
The transcription factor activator protein 1 (AP-1) component protein, phospho-cJun, increased in nuclear extracts from chloramphenicol-induced H1299 cells in both a dosedependent (Fig. 6A) a and time-dependent manner (Supplementary fig. 6). MMP-13 expression could be repressed by the AP-1 inhibitor curcumin (Fig. 6B), but not by the nuclear factor-jB inhibitor pyrrolidine dithiocarbamate or Bay-17-7085 (Supplementary fig. 4). The direct DNA binding between phospho-cJun protein and AP-1 consensus sequence (TGACTCA) were demonstrated by EMSA studies in vitro (data not shown).
ChIP was used for assaying the phospho-cJun proteinbinding activity to the MMP-13 promoter. Validation of the method is shown in Supplementary figure 5. The phospho-cJun protein was coimmunoprecipitated together with the MMP-13 promoter in both a dose-dependent (Fig. 6C) and a timedependent manner (Supplementary fig. 6). Coincubation with SP 600125 or LY 294002 inhibited the interaction between phospho-cJun and MMP-13 promoter (Fig. 6D). This finding suggests that the transcription factor AP-1 is required for chloramphenicol-induced MMP-13 expression and further MMP-13-mediated cell invasion.

Antibiotics Causing Mitochondrial Stress and ATP Restriction Induced MMP-13 Expression
In addition to chloramphenicol, other antibiotics, including minocycline, doxycycline, and clindamycin, may also inhibit 146 mitochondrial translation (Fig. 7A), cause ATP restriction (Fig. 7B), induce MMP-13 mRNA expression (Fig. 7C), and facilitate cell invasion (Fig. 7D). It has been reported previously that doxycycline inhibits MMPs. For MMP-13, doxycycline did not function as an inhibitor below 200lM (48 lg/ml). The inhibition concentration may be difficult to achieve in vivo (Smith et al., 1998). However, the 10 lg/ml doxycycline induced MMP-13 expression in H1299 cell.

DISCUSSION
Tumor cells usually change their mitochondrial function, triggers a mitochondria-to-nucleus signaling, and changes gene expression that make the tumor cells more likely to invade (Fosslien, 2008;Uchiyama et al., 2004). The MMPs are wellknown metastatic factors (Kuzuya and Iguchi, 2003). Tumor metastatic potential correlated with enzymatic degradation of type IV collagen. MMP-13 shows the highest proteolytic activity among the collagenases and could degrade type IV collagen at both a2(IV) and a1(IV) chains. Thus, MMP-13expressing cells often have strong invasive capacities (Knäuper et al., 1997a). Clinical evidence support the fact that MMP-13 appearances is highly relevant to the invasive of squamous cell carcinomas, non-small cell lung cancer, malignant peripheral nerve sheath tumors, and breast tumors (Holtkamp et al., 2007;Hsu et al., 2006;Nil et al., 2004;Zhang et al., 2008). Our previous studies showed that chloramphenicol treatment caused mitochondrial stress and senescence-associated apoptosis resistance in cancer cells (Li et al., 2005). In this study, we further proved that chloramphenicol treatment caused ATP restriction and enhanced cancer cell invasion both in vitro and in vivo. The involvement of MMP-13 was supported by the fact that both MMP-13 protein and mRNA were induced by chloramphenicol treatment. This enhanced invasion was blocked by MMP-13 inhibitors. The activation of proMMP-13 can be regulated by membrane-type MMPs (MMP-14) and stromelysin (MMP-10) (Itoh and Seiki, 2006;Nakamura et al., 1998). In this study, the expression of MMP-14 did not change significantly. However, MMP-10 and MMP-11 were downregulated, suggesting that the invasive effects mentioned above resulted directly from MMP-13 induction. Furthermore, the activated MMP-13 can activate gelatinases and enhance invasion indirectly (Knäuper et al., 1997b). The blot showed that the phospho-cJun was accumulated in nuclear extract of chloramphenicol-treated H1299 cell. The PCNA was used as internal control of nuclear extract. (B) Chloramphenicol-induced MMP-13 expression was inhibited by the transcription factor AP-1 inhibitor, curcumin (10-50lM). The fold changes of MMP-13 mRNA were analyzed by densitometry. (C) The ChIP was used for assay the interaction between phospho-cJun and MMP-13 promoter. In chloramphenicol-treated cell, the ChIP-captured MMP-13 promoter was increased in dose-dependent manner. (D) Coincubation with SP 600125 and LY 294002 decreased the DNA-binding activity of phospho-cJun protein to MMP-13 promoter. These data suggested that JNK, PI-3K/Akt, and AP-1 activities were required for chloramphenicol-induced MMP-13 mRNA induction. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate a statistical difference with the control. # p < 0.05, ## p < 0.01, and ### p < 0.001 indicate a statistical difference with chloramphenicol-treated control.
Mitochondrial stress, by mitochondrial DNA depletion, hypoxia, or aging, resulted in altered energy metabolism and intracellular calcium homeostasis. Mitochondrial stress also induced mitochondria-to-nucleus stress signaling, such as MAPKs and PI-3 kinase/Akt signaling (Uchiyama et al., 2004). The specific stress signaling confirmed apoptosis resistance and enhanced cancer cell invasion (Fosslien, 2008). Recently, 2,3,7,8-tetrachlorodibenzo-p-dioxin has been demonstrated to interfere with mitochondrial transcription, inhibit apoptosis, and lead to invasive phenotype formation (Biswas et al., 2008). In mitochondria-damaged cells, with the increase of cytosolic calcium, calcineurin-sensitive NF-jB/Rel factor is activated. This signaling strengthens the expression of cathepsin L and cathepsin B and leads to an increase in invasive ability (Amuthan et al., 2001;Biswas et al., 2008;Hamer et al., 2009).
In our study, we found that chloramphenicol-induced PI-3K/JNK/AP-1 activation might function as a novel mitochondria-to-nucleus stress signal. The signal increased MMP-13 expression and MMP-13-associated cancer cell invasion. Moreover, antibiotics with the ability to inhibit mitochondrial translation would also cause senescenceassociated apoptosis resistance (Li et al., 2005) and MMP-13 expression. These antibiotics have been used for many years in the treatment of skin and other infections. Hence, the clinical significance of our study is that chloramphenicol application, as well as other 70S ribosomal inhibitors, should be used with caution, especially during cancer therapy. As a result, the chloramphenicol-treated solid-tumor cell also provides a model for further investigation of mitochondrial damage and cancer progression.