Generation and characterization of a serum albumin-humanized mouse model and its utility for detection of albumin adduct formation in vivo.

Abstract

An albumin-humanized mouse model was generated via crossbreeding between albumin-transgenic and albumin-null mice and characterized. Immunoblot analyses confirmed that only human, but not mouse, albumin was expressed, and they revealed that the level of serum human albumin in the albumin-humanized mouse was 5-6 times the level in the albumin-transgenic mouse. Serum albumin levels (measured in plasma) were 3-4 times lower, whereas globulin levels were 2 times higher, in the albumin-humanized than in wild-type mice. The transgene integration site was localized via optical genome mapping to mouse chromosome 14, between 2 marker positions that are 143-kilobase pairs apart. A neighboring gene (alpha fetal protein) to albumin on the human chromosome was confirmed absent in the transgenic mouse. A transgene-mouse genome junction sequence was further identified, enabling a new protocol to distinguish between hemizygous and homozygous albumin-transgenic mice. Transgenic human albumin formed covalent adducts with naphthalene 1,2-oxide (NAO) ex vivo at the Cys³⁴ residue, which was recovered in the peptide ³¹LQQCPF³⁶ following tryptic/chymotryptic digestion. LQQC∗^[NAO]PF, structurally confirmed by mass spectral comparisons with synthetic adduct standards, was also detected in protease-digested plasma from homozygous albumin-humanized mice that were exposed to naphthalene through intraperitoneal injection (200 mg/kg) or inhalation (20 ppm). Pharmacokinetic analysis of plasma NAO-albumin, in comparison with NAO-glutathione, revealed a later time at maximal concentration (9.6 versus 2 hours), lower maximal concentration (0.21 versus 12 μM), and longer elimination half-life (79.7 versus 2.3 hours) for NAO-albumin following intraperitoneal naphthalene injection; the persistence of NAO-albumin adduct was also demonstrated following inhalation naphthalene exposures. SIGNIFICANCE STATEMENT: An albumin-humanized mouse was generated and characterized to demonstrate its utility for detecting in vivo formation of reactive metabolite adducts with human albumin. Key findings, including detection of the albumin adduct with naphthalene 1,2-oxide (NAO) at Cys³⁴ following exposure of mice to naphthalene, a ubiquitous pollutant and possible human carcinogen, and persistence of the NAO-albumin adduct in plasma relative to NAO-glutathione adducts, indicate that the model will be valuable for discovering biomarkers of exposure and potential toxicity for numerous drugs and other xenobiotics.