BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2

doi:10.1016/j.freeradbiomed.2006.01.025

Free Radical Biology and Medicine

Volume 40, Issue 10, 15 May 2006, Pages 1843-1856

https://doi.org/10.1016/j.freeradbiomed.2006.01.025 Get rights and content

Abstract

NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1^−/− with NQO2^−/− mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial-associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFα, IL-6, and IL-1β and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury.

Section snippets

Generation and genotyping of DKO mice

NQO1^−/− mice were cross-bred with NQO2^−/− mice to generate heterozygous NQO1^+/−/NQO2^+/− mice. The heterozygous animals were interbred to produce homozygous DKO mice. This process deleted exon 6 of NQO1 and exon 3 of NQO2 and replaced them with 2-kb neo cassettes in the DKO mice (Fig. 1A). The mice were kept in a pathogen-free environment in polycarbonate cages, maintained with a 12-h light/dark cycle, a temperature of 24 ± 2°C, a relative humidity of 55 ± 10%, and a negative atmospheric

Generation and characterization of DKO mice

The cross-breeding of NQO1^−/− with NQO2^−/− mice led to successful generation of DKO mice. The wild-type and DKO alleles are shown in Fig. 1A. Exon 6 of the NQO1 and exon 3 of the NQO2 gene were deleted and replaced with the neo cassette. The tail DNA from wild-type, NQO1^−/−, NQO2^−/−, and DKO mice was analyzed for the presence of wild-type and mutant NQO1 and NQO2 by restriction digestion, Southern blotting, and hybridization (Fig. 1B). The presence of wild-type and mutant NQO1 and NQO2 alleles

Discussion

Several lines of evidence indicate that we successfully generated double-knockout NQO1^−/−/NQO2^−/− mice deficient in expression of both NQO1 and NQO2. The genotyping by Southern blotting and genomic PCR and hybridization revealed that exon 6 of NQO1 and exon 3 of NQO2 were replaced with the neo cassette, leading to inactivation of these genes in DKO mice. NQO1 and NQO2 mRNAs and proteins were not detected in DKO mice. NQO1 and NQO2 activities were absent or present in insignificant amounts in

Acknowledgments

We thank Ms. Namphuong Tran for technical assistance. We are also thankful to Dr. Dorothy Lewis, Baylor College of Medicine (Houston, TX, USA) for help in flow-cytometric analysis. This investigation was supported by NIH Grant RO1 ES07943 to A.K.J. and RO1 HL070921 to B.M.

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The protective effects of CYP1A enzymes against hyperoxia-induced lung injury in rodents have been extensively documented [26,41–43]. In addition, NQO1 has been shown to protect cells and tissues against oxidant injury induced by various toxic chemicals [44] and oxygen [45]. The protective mechanisms of these enzymes have been attributed to their ability to conjugate and scavenge the reactive electrophiles and lipid peroxidation products generated by an oxidant injury [46].
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¹: These authors contributed equally to this work.

²: Present address: Mayo Clinic, GI Research Unit, Alfred 2-435, 200 First Street SW, Rochester, MN 55905, USA.

³: Present address: Biology Division, GVK Biosciences, #210 “My Home Tycoon”, 6-3-1192, Begumpet, Hyderabad-500016, India.

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Original ContributionBALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2

Abstract

Section snippets

Generation and genotyping of DKO mice

Generation and characterization of DKO mice

Discussion

Acknowledgments

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood

Blood

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Immunol. Today

Quinone reductases multitasking in the metabolic world

Drug Metab. Rev.

Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase)

Proc. Natl. Acad. Sci. USA

Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy

Cancer Res.

Disruption of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene in mice causes myelogenous hyperplasia

Cancer Res.

NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis

Cancer Res.

NAD(P)H:quinone oxidoreductase 1 deficiency and increased susceptibility to 7,12-dimethylbenz[a]anthracene-induced carcinogenesis in mouse skin

J. Natl. Cancer Inst.

Localization of human NQO1 gene to chromosome 16q22 and NQO2—6p25 and associated polymorphisms

Pharmacogenetics

Original Contribution
BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2