Up-regulation of xenobiotic detoxification genes and increased xenobiotic resistance in Little mice
An elevation of xenobiotic metabolism has been recently proposed as a mechanism of extended longevity. The main hypothesis is that the molecular and cellular damage produced by the toxic compounds that this system targets, in particular toxic lipophilic by-products of metabolism, is a major contributor to the aging process (Gems & McElwee, 2005). Here, we investigated the alterations in xenobiotic metabolism in the long-lived Little mice.
These mutant mice showed a remarkably strong and concerted transcriptional up-regulation of xenobiotic detoxification genes. We found that this transcriptional up-regulation was associated with significant in vivo alterations in xenobiotic resistance. Little mice displayed a potent increase in resistance against the adverse effects of the hepatotoxins acetaminophen and bromobenzene and against the paralyzing agent zoxazolamine. The increased resistance against zoxazolamine can be explained by the up-regulation of cytochromes p450s in Little mice, because the rate of zoxazolamine inactivation directly reflects cytochrome p450 activity.
The basis for acetaminophen toxicity resides largely in its conversion to the highly reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) that covalently binds to cellular macromolecules. NAPQI is normally detoxified by glutathione S-transferases through conjugation with glutathione (GSH). However, NAPQI can accumulate if its rate of production exceeds the rate of GSH-conjugation or when GSH levels are depleted (Hinson et al., 2004). The increased expression of many glutathione S-transferase genes in Little mice, which should rapidly detoxify NAPQI after its formation from acetaminophen, likely contributes to the increased resistance against acetaminophen-toxicity in these mice. This agrees with previous studies that show that pharmacological inducers of phase II enzymes, in particular glutathione S-transferases, protect against acetaminophen hepatotoxicity in mice (Ansher et al., 1983; Seo et al., 2000).
Although Little mice up-regulate cytochrome p450s, and therefore are also expected to produce the toxic intermediate (NAPQI) at a higher rate, the resistance of Little mice against this compound suggests that the up-regulation of glutathione S-transferases increases the rate of conjugation (detoxification) to compensate for increased NAPQI production. This situation could potentially lead to the depletion of glutathione; however, besides conjugation with glutathione, acetaminophen can also be detoxified by sulfation or glucuronidation to form acetaminophen-sulfate and acetaminophen-glucuronide (Brouwer & Jones, 1990; Zamek-Gliszczynski et al., 2006). The increased expression of various sulfotransferases (Sth2, Sult1a1, Sult1d1, and Sult1e1) and the glucuronosyltransferase Ugt1a1 in Little mice could increase their capacity for acetaminophen sulfation and glucuronidation and likely contributes to their enhanced resistance against this compound and would also ameliorate glutathione depletion by enhancing this alternative route of acetaminophen detoxification. Extensive sulfation can cause a depletion of the universal sulfonate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS). Importantly, Little mice presented a significant up-regulation of Papss2 (3′-phosphoadenosine 5′-phosphosulfate synthase 2), which is responsible for the biosynthesis of the sulfate donor PAPS in mouse liver. Other mouse models of extended longevity have also shown increased resistance against acetaminophen-induced toxicity, such as calorie-restricted mice and methionine-restricted mice (Miller et al., 2005; Harper et al., 2006).
Bromobenzene toxicity depends on its conversion by cytochrome p450s to a toxic intermediate (bromobenzene 3,4-epoxide) that is subsequently detoxified by glutathione S-transferases. The resistance of Little mice against this compound should share a similar basis as with their resistance against acetaminophen.
Interestingly, there is some direct experimental evidence for a role of glutathione S-transferases in longevity. Overexpression of C. elegans gst-10 or mouse Gsta4 increases stress resistance and lifespan in C. elegans (Ayyadevara et al., 2005). Both of these GSTs have high catalytic efficiency toward the conjugation of the lipid peroxidation product 4-hydroxynonenal (4-HNE), which is known to accumulate with age in diverse systems (Sohal & Weindruch, 1996; Levine & Stadtman, 2001; Sohal et al., 2002; Zheng et al., 2005). The up-regulation of Gsta4 in Little mice suggests that the elevation of glutathione S-transferase activity may be a mechanism of longevity assurance shared between nematodes and mice.
In contrast with the increased resistance to acetaminophen and bromobenzene-induced toxicity, we found that Little mice were more sensitive to carbon tetrachloride-induced hepatotoxicity. The toxic effects of CCl4 come from its activation by cytochrome p450s to form trichloromethyl free radical (CCl3), which readily reacts with oxygen to form CCl3O2. These highly reactive species initiate cellular damage by covalent binding to macromolecules and by inducing lipid peroxidation (Weber et al., 2003). Interestingly, although the mechanisms of CCl4 toxicity have been studied extensively, a role for glutathione S-transferases or sulfotransferases in the detoxification of its toxic intermediates has not been described (Weber et al., 2003) and some studies suggest that the CCl4 free radical products are unlikely to be detoxified by a GSH-dependent mechanism (Burk et al., 1984). Therefore, the increased sensitivity of Little mice to CCl4-induced hepatotoxicity must be a consequence of their elevation in cytochrome p450s. In this case, however, in contrast with the two previously discussed hepatotoxins, the elevation in glutathione S-transferases or sulfotransferases cannot provide a protective effect.
The classic xenobiotic receptors Car and Pxr are key regulators of all phases of xenobiotic metabolism and several of their target genes are up-regulated in Little mice. However, the results of our experiments using the double mutants Car/Little and Pxr/Little and the triple mutant Car/Pxr/Little demonstrate that, with the exception of Cyp2b10 and Cyp2c38, these xenobiotic nuclear receptors are not required for the up-regulation of xenobiotic genes in Little mice. There are a number of other nuclear receptors and transcription factors that could be involved in this up-regulation, including Aryl hydrocarbon receptor (Ahr), Peroxisome proliferator activated receptors (Ppar), Vitamin D receptor (Vdr), NF-E2-related factor-2 (Nrf2), and others (Wang & LeCluyse, 2003; Xu et al., 2005). The Nrf2 transcription factor is an interesting candidate. Hepatocyte-specific deletion of the Nrf2-repressor Keap1 constitutively activates Nrf2 and up-regulates a set of xenobiotic genes that partially overlaps with the genes up-regulated in Little mice. These overlapping genes include Sth2, Cyp2b13, Fmo3, Cyp2b9, Gstm3, Mgst3, and Gsta2 (Okawa et al., 2006). Additionally, like Little mice, the Keap1 mice have increased resistance against acetaminophen-induced hepatotoxicity (Okawa et al., 2006).
We found that the Little mutation reverses the susceptibility of the Car mice against zoxazolamine induced-paralysis, and the susceptibility of the Car/Pxr mice against acetaminophen-induced liver necrosis. It has been previously reported that loss of Car function significantly increases zoxazolamine sensitivity (Wei et al., 2000). The constitutive up-regulation of cytochrome p450s introduced by the Little mutation is likely to be responsible for the loss of sensitivity against zoxazolamine-induced paralysis in the Car/Little double mutant mice. We found that the combined loss of Car and Pxr results in considerably increased susceptibility to acetaminophen-induced hepatotoxicity. Although at this time the precise mechanisms for the increased toxicity in Car/Pxr mice are unclear, they may be related to the failure to activate key phase I or phase II xenobiotic detoxification genes. Most likely, the constitutive up-regulation of xenobiotic detoxification genes associated with the introduction of the Little mutation is responsible for reversing this susceptibility in the Car/Pxr/Little mice.
Bile acids as potential mediators of the xenobiotic response in Little mice
Little mice showed significantly elevated bile acid levels in bile, liver, and plasma. Our data indicated that this elevation was primarily a consequence of a generalized increase of bile acid synthesis, because the fecal bile acid excretion rate, which is directly proportional to the amount synthesized in the liver, was considerably elevated in Little mice. We found that the expression of the canalicular bile salt export pump (Bsep), the major bile salt export pump in rodents, and Mrp2, another major bile salt export pump, were not affected in Little mice implying that bile acid export was not compromised. However, we observed some alterations in genes involved in bile acid uptake by the liver. The Na+-independent organic anion-transporting polypeptide Slc21a1, important for bile salt uptake into hepatocytes, was greatly down-regulated in Little mice and this may contribute to the elevation in plasma bile acids levels. This down-regulation may constitute a defense response against the elevation of liver bile acids because Slc21a1 was also down-regulated in the CA-treated wild-type mice. Interestingly, Slc21a5, which shows overlapping substrate specificities to Slc21a1, is highly up-regulated in Little mice and in the CA-treated wild-type mice. It has been proposed that Slc21a5 plays an important role in situations in which the expression of Slc21a1 is compromised and its up-regulation may represent an important response in the hepatic detoxification of bile acids (Meier & Stieger, 2002). Although Little mice could be regarded as mildly cholestatic, the elevation of bile acids in Little mice does not induce a pathological state of the liver. Little mice do not show histological signs of liver damage and their ALT and LDH serum levels are normal.
The analysis of biliary bile acids showed an almost four-fold elevation in the levels of CA, which also resulted in an altered bile acid composition in Little mice with a two-fold increase in the proportion (as a percentage of total bile acids) of this bile acid. We found that CA treatment of wild-type mice up-regulated a largely overlapping set (close to 80%) of the xenobiotic genes up-regulated in Little mice. These observations support the idea that bile acids, in particular CA, play a role in the up-regulation of these genes in Little mice. Previously, CA has been shown to up-regulate Cyp2b10, Cyp3a11 and affect the expression of bile acid transporters (Fickert et al., 2001; Zollner et al., 2006), but such a broad response on the expression of phase I and phase II detoxification genes has not, to our knowledge, been documented before.
Some of the genes up-regulated in CA-treated wild-type mice do not reach the levels seen in Little mice. This may be due to the fact that the elevation in bile acids in Little mice is a chronic state vs. the acute nature of CA treatment or it may be an indication that other mechanisms (including the elevation in other bile acids) also play a role in the induction of these genes in Little mice. Treatment of Little mice with CA yielded some interesting observations. Little mice, just like wild-type mice, decreased the expression of the bile acid synthesis genes Cyp7a1 and Cyp8b1, and up-regulated the nuclear receptor Shp after CA treatment. This indicated that the transcriptional mechanisms of negative feedback regulation of bile acid synthesis, which are largely dependent on Fxr, are functional in Little mice. Because the basal expression levels of these genes are not affected in Little mice, it is possible that they have a higher threshold for the activation of these negative feedback mechanisms than normal mice. The two- or three-fold elevation of bile acids in these mutant mice may not be enough to activate this response, but when there is a very high or sudden elevation in bile acid levels, as with cholic acid treatment, the response can be triggered.
We are proposing that the elevation of bile acid levels, and in particular CA levels, is a likely contributor to the increased expression of xenobiotic detoxification genes and increased xenobiotic resistance in Little mice. However, the elevation of other endobiotics (some of them may be other bile acids) could also contribute. Furthermore, in addition to the proposed contribution of bile acids to the elevation of xenobiotic detoxification genes, it is possible that the simple increase in bile acid excretion rates, by itself, may be an important contributor to the increased xenobiotic resistance in Little mice. Besides renal excretion, biliary excretion is a major mechanism involved in the elimination of xenobiotics, and an increase in the rate at which conjugated xenobiotics are excreted into bile is expected to decrease their toxicity. Of particular relevance to the increased resistance against acetaminophen and bromobenzene in Little mice, sulfate and glucuronide metabolites of acetaminophen are known to be excreted into bile (Brouwer & Jones, 1990; Zamek-Gliszczynski et al., 2006), as well as bromobenzene-glutathione conjugates (Madhu & Klaassen, 1992).
Our analysis of the Fxr/Little double mutant mice further confirmed a dysregulation of bile acid homeostasis in Little mice. We observed a remarkable synergistic effect in the elevation of bile acid levels in the Fxr/Little mice. This dramatic elevation in bile acid levels occurred without a significant up-regulation of Cyp7a1 in the Fxr/Little mice as compared to either wild-type or Fxr mice (Table 8). However, the expression of canalicular Bsep is severely decreased in Fxr mice, and this down-regulation, not present in Little mice, is maintained in the Fxr/Little mice (Table 8). It is likely that the combination of the down-regulation of Bsep in Fxr mice with the increased bile acid biosythesis in Little mice is a primary cause for the accumulation and elevation of bile acid levels in these double mutants. The liver damage seen in the Fxr/Little mice is very likely a consequence of bile acid accumulation and toxicity. In particular, the prominent bile ducts seen in the livers of the Fxr/Little mice are likely to be related to the elevation in bile acid levels, because it has been previously shown that bile acid feeding enhances bile duct proliferation while external bile drainage decreases proliferation (Alpini et al., 1999; Alvaro et al., 2000).
Importantly, many of the xenobiotic detoxification genes up-regulated in Little mice lost completely, or to some extent, their elevated levels of expression in the Fxr/Little mice. The majority of these genes are not known targets, direct or indirect, of Fxr. It is possible that these genes represent previously unidentified Fxr targets, and that the loss of their up-regulation contributes to the accumulation of bile acids and to the liver damage phenotype of the Fxr/Little mice. These observations support a model in which bile acids, through the activation of Fxr, up-regulate a large portion of the xenobiotic detoxification genes in Little mice. As mentioned before, Fxr plays a central role in bile acid homeostasis by regulating bile acid synthesis genes and bile acid transporters. The regulation by Fxr of xenobiotic genes involved in bile acid detoxification would be consistent with this role and would add another level to the regulation of bile acid homeostasis by this nuclear receptor. However, there is a possibility that the down-regulation of these genes in the Fxr/Little mice could be a nonspecific consequence of liver damage and hepatotoxicity and further investigations are required to clarify this issue.
Our results support the hypothesis that alterations in xenobiotic metabolism and increased xenobiotic resistance is a trait associated with longevity in mice. Our analysis leads us to propose that bile acids, possibly acting through Fxr, may be important mediators of this response. The next step to further these hypotheses would be to test the effects that a chronic (possibly lifelong) elevation of xenobiotic detoxification genes would have on aging and longevity. This could be achieved through chemical intervention by using drugs (we would propose the use of bile acid-like compounds) that induce expression of xenobiotic detoxification genes, through the use of additional mouse models with a constitutive elevation of xenobiotic detoxification genes such as the Keap1 knockout mice (Okawa et al., 2006) or by genetic interventions that involve the overexpression of constitutively active forms of nuclear receptors (we would propose Fxr as an interesting candidate) that regulate the xenobiotic response.