Increased Nrf2 activation in livers from Keap1-knockdown mice increases expression of cytoprotective genes that detoxify electrophiles more than those that detoxify reactive oxygen species

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance-associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.

FIG. 1.

Illustration of oxidative and electrophilic stress. Oxidative stress is represented on the left side and electrophilic stress on the right side. Xenobiotics can undergo redox cycling, such as in the case of paraquat, which generates superoxide in a reaction catalyzed by cytochrome p-450 reductase (Cpr). Detoxificaton of superoxide (O₂^−.) to hydrogen peroxide (HOOH) is mediated by Sod. HOOH is detoxified to H₂O by Cat, Prx, and Gpx. GSH is oxidized to glutathione disulfide (GSSG) in the Gpx-catalyzed reaction. If HOOH is not detoxified, it can undergo a Fenton reaction to generate hydroxyl radical (OH^.). O₂^−. can also react with nitric oxide (NO^.) to form peroxynitrite (ONOO⁻), which can be detoxified by Gpx, Prx, or GSH. Biotransformaton of xenobiotics by Cyps and Pod can lead to the formation of nucleophiles or electrophiles. Detoxification of electrophilic intermediates by Nqo1 or Eh-1 creates nucleophiles, which can be conjugated with glucuronic acid via Ugts or conjugated with sulfate via Sults and excreted by Mrps. Electrophiles can also be conjugated with GSH in a reaction catalyzed by Gsts and excreted via Mrps. GSH and its synthesis play important roles in both superoxide and electrophile detoxification. The rate-limiting enzymes in GSH synthesis are glutamate-cysteine ligase catalytic and modifier subunits (Gclc and Gclm, respectively).

FIG. 2.

Nrf2 protein expression in hepatic nuclear fractions in wild-type, Nrf2-null, and Keap1-kd mice (n = 5) (A). Intensity of protein bands was quantified, and individual blot densities were normalized to wild-type and expressed as mean ± SEM. Messenger RNA expression of Nrf2 and Keap1 in wild-type, Nrf2-null, and Keap1-kd mice (n = 5) (B). Values are expressed as mean ± SEM Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 3.

Hepatic reduced liver GSH content in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 4.

Messenger RNA expression of prototypical Nrf2 targets (A), superoxide and hydrogen peroxide reducing enzymes (B), redoxins (C), and NADPH generating enzymes (D) in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 5.

Messenger RNA expression (A) and enzyme activity (B) of cytochrome p450s in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 6.

Messenger RNA expression of Aldhs (A) and carboxylesterases (B) in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 7.

Messenger RNA expression of Ugts (A) and Gsts (B) in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).

FIG. 8.

Messenger RNA expression of uptake (A) and efflux (B) transporters in wild-type, Nrf2-null, and Keap1-kd mice (n = 5). Values are expressed as mean ± SEM. Asterisks (*) indicate a statistically significant difference from wild-type mice (p ≤ 0.05).