Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation

Abstract

Tamoxifen (TAM) is commonly used for cell type specific Cre recombinase-induced gene inactivation and in cell fate tracing studies. Inducing a gene knockout by TAM and using non-TAM exposed mice as controls lead to a situation where differences are interpreted as consequences of the gene knockout but in reality result from TAM-induced changes in hepatic metabolism. The degree to which TAM may compromise the interpretation of animal experiments with inducible gene expression still has to be elucidated. Here, we report that TAM strongly attenuates CCl-induced hepatotoxicity in male C57Bl/6N mice, even after a 10 days TAM exposure-free period. TAM decreased (p < 0.0001) the necrosis index and the level of aspartate- and alanine transaminases in CCl-treated compared to vehicle-exposed mice. TAM pretreatment also led to the downregulation of CYP2E1 (p = 0.0045) in mouse liver tissue, and lowered its activity in CYP2E1 expressing HepG2 cell line. Furthermore, TAM increased the level of the antioxidant ascorbate, catalase, SOD2, and methionine, as well as phase II metabolizing enzymes GSTM1 and UGT1A1 in CCl-treated livers. Finally, we found that TAM increased the presence of resident macrophages and recruitment of immune cells in necrotic areas of the livers as indicated by F4/80 and CD45 staining. In conclusion, we reveal that TAM increases liver resistance to CCl-induced toxicity. This finding is of high relevance for studies using the tamoxifen-inducible expression system particularly if this system is used in combination with hepatotoxic compounds such as CCl.

Dissecting the Role of Subtypes of Gastrointestinal Vagal Afferents

“…On the other hand, a tetracycline analogue (doxycycline) can be administered via oral (Saam and Gordon, 1999;Lindeberg et al, 2002), intraperitoneal and local injections (Utomo et al, 1999). Further, it is important to note that tamoxifen and doxycycline may introduce confounding factors that should be considered in designing experiment (Moullan et al, 2015;Hammad et al, 2018).…”

Tamoxifen affects chronic pancreatitis‐related fibrogenesis in an experimental mouse model: an effect beyond Cre recombination

“…With respect to immune cells, tamoxifen can result in reduced macrophage infiltration and proinflammatory cytokine expression in male mice , impaired dendritic cell (DC) differentiation, and reduction in the immunostimulatory capacity of DCs (sex not specified) . On the other hand, increased recruitment of Kupffer cells and leukocytes to damaged regions of tamoxifen‐pretreated livers of male mice was accompanied by complex changes in the expression of cytokines and immune‐related genes . Proinflammatory cytokine production was found to be either enhanced or dampened by ER activity .…”

“…injections, oral gavage, or topical application (skin) of tamoxifen on one or several consecutive days. This is followed by varied drug-free days after the last tamoxifen application in order to allow for optimal recombination before further interventions [4][5][6]. However, recent studies indicated that tamoxifen not only exerts the role of activating Cre recombinase, but also has various side effects in different genetically engineered mouse models [4,5,7].…”

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society

“…A single dose of tamoxifen is reported to have irreversible effects on the uterus. Furthermore, tamoxifen crosses the blood brain barrier to regulate energy metabolism, is a potent immune modulator in mice, and can induce bone marrow failure (205)(206)(207). Investigators who choose to use tamoxifen in spite of its potent estrogen receptormediated actions, should be advised to include a no treatment control as well as a tamoxifen-treated/no Cre control.…”