Alterations in xenobiotic metabolism in the long-lived Little mice

Abstract

Our previous microarray expression analysis of the long-lived Little mice (Ghrhr(lit/lit)) showed a concerted up-regulation of xenobiotic detoxification genes. Here, we show that this up-regulation is associated with a potent increase in resistance against the adverse effects of a variety of xenobiotics, including the hepatotoxins acetaminophen and bromobenzene and the paralyzing agent zoxazolamine. The classic xenobiotic receptors Car (Constitutive Androstane Receptor) and Pxr (Pregnane X Receptor) are considered key regulators of xenobiotic metabolism. Using double and triple knockout/mutant mouse models we found, however, that Car and Pxr are not required for the up-regulation of xenobiotic genes in Little mice. Our results suggest instead that bile acids and the primary bile acid receptor Fxr (farnesoid X receptor) are likely mediators of the up-regulation of xenobiotic detoxification genes in Little mice. Bile acid levels are considerably elevated in the bile, serum, and liver of Little mice. We found that treatment of wild-type animals with cholic acid, one of the major bile acids elevated in Little mice, mimics in large part the up-regulation of xenobiotic detoxification genes observed in Little mice. Additionally, the loss of Fxr had a major effect on the expression of the xenobiotic detoxification genes up-regulated in Little mice. A large fraction of these genes lost or decreased their high expression levels in double mutant mice for Fxr and Ghrhr. The alterations in xenobiotic metabolism in Little mice constitute a form of increased stress resistance and may contribute to the extended longevity of these mice.

Fig. 1

Increased resistance to zoxazolamine induced-paralysis in Little mice. Mice were treated with a single intraperitoneal dose of zoxazolamine (250 mg kg⁻¹). The duration of the paralysis was recorded as the time when the mice were able to right themselves repeatedly. The paralysis time was significantly reduced (P < 0.001) in Little mice (n = 10) compared to wild-type mice (n = 10). The increased susceptibility of the Car mice (n = 3) against zoxazolamine induced-paralysis was significantly reversed (P = 0.001) in the Car/Little mice (n = 7), which showed a paralysis time comparable to Little mice. Each bar represents the mean ± (2 × SE) for each group. The P-values refer to a Student’s t-test between the indicated groups.

Fig. 2

Increased resistance to acetaminophen-induced liver toxicity in Little mice. Mice were administered a 375 mg kg⁻¹ dose of acetaminophen by intraperitoneal injection. (A) After 24 h, liver sections were examined by histological staining (hematoxylin and eosin staining). Liver samples for all treated animals were analyzed and representative histology is shown. While the livers from wild-type animals (n = 10) showed extensive necrosis, only minimal necrosis was observed in Little mice (n = 5). The Car/Pxr double mutants (n = 6) showed even more extensive liver damage than wild-type animals. However, the Car/Pxr/Little (n = 4) mice presented only minimal necrosis that was comparable to the levels seen in Little mice. (B) The necrotic area (as a percentage of total area) was determined for each experimental group as described in the methods section. Little mice and Car/Pxr/Little mice showed a reduced necrotic area as compared to wild-type mice and Car/Pxr mice (P < 0.001 for all comparisons). The Car/Pxr mice showed a substancially larger necrotic area as compared to wild-type mice (P < 0.001). Serum levels of ALT (C) and LDH (D) were measured after 24 h. Little mice and Car/Pxr/Little mice had significantly reduced ALT levels as compared to wild-type mice and Car/Pxr mice (P < 0.02 for all comparisons). Little mice and Car/Pxr/Little mice also had reduced LDH levels as compared to wild-type mice and Car/Pxr mice (P < 0.03 for all comparisons). The Car/Pxr mice showed significantly increased LDH levels as compared to wild-type mice (P < 0.006). Basal Levels of ALT and LDH in Car/Pxr and Car/Pxr/Little mice were indistinguishable from wild-type mice and were not affected by treatment with the PBS vehicle (not shown). In (E), the scatter plot shows the correlation between ALT levels and necrotic areas for the individual mice in all experimental groups. In (B), (C), and (D) bars represents the mean ± (2 × SE) for each group. All P-values refer to a Student’s t-test between the indicated groups.

Fig. 3

Little mice are resistant to bromobenzene-induced liver toxicity. Mice were treated with a 0.36 mL kg⁻¹ dose of bromobenzene by intraperitoneal injection. (A) After 24 h, liver sections were examined by histological staining (hematoxylin and eosin staining). Liver samples for all treated animals were analyzed and representative histology is shown. The livers from wild-type animals (n = 5) showed extensive necrosis (indicated by arrows), but only minimal necrosis was observed in Little mice (n = 5). (B) The necrotic area (as a percentage of total area) was determined for each experimental group as described in the methods section. Little mice showed a significantly reduced necrotic area as compared to wild-type mice (P < 0.001). Serum levels of ALT (C) and LDH (D) were measured 24 h after treatment. Little mice showed significantly reduced levels of both ALT (P < 0.001) and LDH (P < 0.002). In (B), (C), and (D) bars represents the mean ± (2 × SE) for each group. All P-values refer to a Student’s t-test.

Fig. 4

Little mice show increased susceptibility to carbon tetrachloride-induced liver toxicity. Mice were administered a 50 µL kg⁻¹ dose of carbon tetrachloride by intraperitoneal injection. (A) After 24 h, liver sections were examined by histological staining (hematoxylin and eosin staining). Liver samples for all treated animals were analyzed and representative histology is shown. The livers from both wild-type (n = 7) and Little mice (n = 5) showed wide-spread damage, however, the liver damage in Little mice was more extensive. (B) The liver damage areas (as a percentage of total area) were determined for each experimental group as described in the methods section. Little mice showed significantly increased liver damage areas as compared to wild-type mice (P < 0.001). (C) Serum levels of ALT were measured after 24 h and were significantly higher in Little mice (P < 0.02). In (B) and (C) bars represent the mean ± (2 × SE). All P-values shown refer to a Student’s t-test.

Fig. 5

Elevation of bile acid levels and increased bile acid excretion rate in Little mice. Biliary, serum, and liver bile acids were measured in Little mice as described in the experimental procedures. (A) Little mice (n = 5) showed significantly elevated levels of total biliary bile acids (P < 0.02) and biliary cholic acid (P < 0.005) as compared to wild-type mice (n = 5). (B) Little mice (n = 7) also showed significantly elevated levels of total serum bile acids (P < 0.0001), serum cholic acid (P < 0.002), and serum β-muricholic acid (P < 0.02) as compared to wild-type mice (n = 8). (C) Total liver bile acid levels were also significantly elevated (P < 0.0001) in Little mice (n = 25) as compared to wild-type mice (n = 25). (C) Bile acid excretion rates were determined as described in the experimental procedures. Little mice (n = 5) had a significantly higher bile acid excretion rate (P < 0.0001) than the wild-type mice (n = 7). P-values shown refer to a Student’s t-test, bars represents the mean ± (2 × SE).

Fig. 6

Liver histology and elevation of bile acids in the Fxr/Little mice. (A) An histological examination (hematoxylin and eosin staining) of the livers extracted from these mice revealed extensive damage and morphologic alterations, including steatosis, isolated necrosis, prominent and proliferating bile ducts, and focal inflammation. (B) Liver bile acid levels were over 10-fold elevated in the Fxr/Little (n = 4) mice as compared to wild-type mice (n = 25) (P < 0.0001) and over five-fold elevated as compared to Little mice (n = 25) or Fxr mice (n = 9) (P < 0.0001 for both comparisons). (C) Serum bile acid levels were over 150-fold elevated in the Fxr/Little mice as compared to wild-type mice (P < 0.0001) and over 50-fold elevated as compared to Little mice or Fxr mice (P < 0.0001 for both comparisons). P-values shown refer to a Student’s t-test, bars represents the mean ± (2 × SE).