Biochemical Properties of Atranorin-Induced Behavioral and Systematic Changes of Laboratory Rats

Abstract

Atranorin (ATR) is a secondary metabolite of lichens. While previous studies investigated the effects of this substance predominantly in an in vitro environment, in our study we investigated the basic physicochemical properties, the binding affinity to human serum albumin (HSA), basic pharmacokinetics, and, mainly, on the systematic effects of ATR in vivo. Sporadic studies describe its effects during, predominantly, cancer. This project is original in terms of testing the efficacy of ATR on a healthy organism, where we can possibly attribute negative effects directly to ATR and not to the disease. For the experiment, 24 Sprague Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided into four groups. The first group (n = 6) included healthy males as control intact rats (♂INT) and the second group (n = 6) included healthy females as control intact rats (♀INT). Groups three and four (♂ATR/n = 6 and ♀ATR/n = 6) consisted of animals with daily administered ATR (10mg/kg body weight) in an ethanol-water solution per os for a one-month period. Our results demonstrate that ATR binds to HSA near the binding site TRP214 and acts on a systemic level. ATR caused mild anemia during the treatment. However, based on the levels of hepatic enzymes in the blood (ALT, ALP, or bilirubin levels), thiobarbituric acid reactive substances (TBARS), or liver histology, no impact on liver was recorded. Significantly increased creatinine and lactate dehydrogenase levels together with increased defecation activity during behavioral testing may indicate the anabolic effect of ATR in skeletal muscles. Interestingly, ATR changed some forms of behavior. ATR at a dose of 10 mg/kg body weight is non-toxic and, therefore, could be used in further research.

Keywords: atranorin; behavioral changes; human serum albumin; laboratory rats; metabolomics; microsomal stability.

Figure 1

3D model of atranorin structure. The white color indicates an atom of hydrogen (H) and the red color indicates an atom of oxygen (O).

Figure 2

Binding of atranorin near the binding site TRP214 in human serum albumin (data from docking analysis).

Figure 3

Representative MRM ion-chromatograms of atranorin (20 ng/mL).

Figure 4

Body mass gain in male (A) and female (B) animals in INT (healthy intact) and ATR (atranorin) groups. Data are expressed as mean ± standard deviation (SD). Significance vs. INT is given as * p < 0.05; ** p < 0.01, and *** p < 0.001, respectively.

Figure 5

Representative photomicrographs of liver histopathology (200×): (A) liver of INT control rats showing normal histology; (B) liver of rats after oral administration of 10 mg/kg b.w. of ATR showing normal liver histology.

Figure 6

(a) Partial least squares-discrimination analysis (PLS-DA) of selected metabolites in ♂INT, ♂ATR, and ♀INT and ♀ATR animals. In the graphical output, 95% confidence ellipses for specific groups are included; (b) variable importance in projection (VIP) plot, calculated from PLS-DA method, displays the top 15 most important metabolite features identified by PLS-DA. Boxes on the right indicate the relative concentration of the corresponding metabolite in the blood in descending order of importance. VIP is a weighted sum of squares of the PLS-DA loadings considering the amount of explained Y-variable in each dimension. The most important features have VIP values of >2.0.

Figure 7

(a) Partial least squares-discrimination analysis (PLS-DA) of selected metabolites in INT and ATR animals and intact female. In the graphical output, 95% confidence ellipses for specific groups are included; (b) variable importance in projection (VIP) plot, calculated from PLS-DA method, displays the top 10 most important metabolite features identified by PLS-DA. Boxes on the right indicate the relative concentration of the corresponding metabolite in the blood in descending order of importance. VIP is a weighted sum of squares of the PLS-DA loadings considering the amount of explained Y-variable in each dimension. The most important features have VIP values of >2.0.