DETERMINATION OF USNIC ACID EFFECT ON IRON METABOLISM IN RAT LIVER

Analysis of hepatic iron content was measured by inductively coupled plasma mass spectrometry. Results and discussion. The results showed that treatment of Fischer 344 (F344) rats with 60 mg usnic acid/kg bw/day for 6 weeks increased the expression of iron metabolism-related genes, Ftl and Fth , while the level of Tfrc mRNA was decreased in rat liver. Additionally, exposure of rats to usnic acid resulted in decrease of hepatic cellular iron content. Conclusion. The obtained results demonstrated that the exposure of rats to a low dose of usnic acid resulted in changes in the expression of iron metabolism related genes. homeostasis may exacerbate liver injury induced by hepatotoxicants [14]. The results of present study demonstrate that administration of UA led to changes in the expression of iron exchange genes in rat liver, especially an up-regulation of Ftl, Fth genes and a down-regulation of Tfrc.

A wide range of environmental toxic substances causes adverse effects on organism and may represent a serious threat to human health. Exposure to xenobiotics may cause a broad range of metabolic disturbances, including iron homeostasis. Iron is both an essential trace element to the body and potentially toxic substance in excess. Perturbations in iron metabolism relate to the growing of various pathological states, including liver toxicity development and progression. Liver is the most important organ for a metabolic process and detoxification of harmful substances, along with iron storage and homeostasis.
Usnic acid is a well-studied secondary metabolite isolated from lichens, which is extensively studied the broad variety of biological features. It is reported that usnic acid is effective for a range of pharmacological purposes such as antiviral, antimicrobial, antiproliferative, antiprotozoal, antitumor and anti-inflammatory. (+)-Usnic acid has been marketed as an ingredient of dietary supplements to promote weight loss. However, some studies reported that usnic acid has been associated with clinical cases of liver toxicity and contact allergy depending on the doses, thus its potential use as a drug is limited.
However, there are limited data on the impact of usnic acid on iron homeostasis in the liver. The purpose of the present study was to investigate the possible effect of usnic acid on iron metabolism in rat liver.
Material and methods. Male Fischer 344 rats (6 weeks of age) were housed in sterilized cages in a temperature-controlled room (24 °C) with a 12 h light/ dark cycle, and given ad libitum access to water and NIH-41 irradiated pelleted diet. Gene expression in the liver of rats was analyzed by quantitative reverse transcription polymerase chain reaction techniques. Analysis of hepatic iron content was measured by inductively coupled plasma mass spectrometry.
Results and discussion. The results showed that treatment of Fischer 344 (F344) rats with 60 mg usnic acid/kg bw/day for 6 weeks increased the expression of iron metabolism-related genes, Ftl and Fth, while the level of Tfrc mRNA was decreased in rat liver. Additionally, exposure of rats to usnic acid resulted in decrease of hepatic cellular iron content.
Conclusion. The obtained results demonstrated that the exposure of rats to a low dose of usnic acid resulted in changes in the expression of iron metabolism related genes.
Research relation to the programs, plans, and department themes. The work is a fragment of research work «Development of methods of diagnosis, treatment and prevention of dental diseases in the population living in environmentally unfavorable conditions», the number of state registration is 011U003681. The study was partially conducted at the National Center for Toxicological Research, USA.
Introduction. Usnic acid (C 18 H 16 O 7 , UA) is a prominent secondary metabolite, that has been used for various purposes worldwide, found in lichensespecially abundant in the Alectoria, Cladonia, Evernia, Lecanora, Ramalina and Usnea genera [1,2]. UA found in two enantiomeric forms: (-) L-usnic acid and (+) D-usnic acid, indicating an R or S projection of the methyl group at position 9b. Both enantiomers possess a broad spectrum of different biological activities [3].
The cumulative data show that UA have been used as traditional medicines for antimicrobial, antiprotozoal, antimycotic, antiviral, antiproliferative, antioxidation, analgesic, antipyretic, anti-inflammatory, wound-healing purposes, chelation of heavy metals, antitumor effects in different cell types and UV light protection [1][2][3]. Moreover, UA is used as an ingredient of powder, toothpaste, sunscreens, deodorants, cream, hair shampoos, and mouthwash [1]. During the past decade, UA has been also used for weight loss as a component of some dietary supplements because of its ability to increase fat metabolism and to raise basal metabolic rate [3,4]. However, it has been reported that dietary supplements containing UA are associated with acute liver failure and toxicity [5,6].

Ivano-Frankivsk National Medical University, Ukraine
Irakindrat0603@gmail.com broad range of important physiological and molecular processes [9]. Among the known proteins that regulate iron homeostasis, expression of Tfrc, Ftl, Fpn1 and Fth -iron metabolism-related genes, are the main ones involved in iron homeostasis changes. In particular, Tfrc gene is known to encode the transferrin receptor 1, which is responsible for iron import from transferrin into cells by endocytosis [15]. Changes in expression of these proteins induced by xenobiotics could lead to an impaired iron metabolism system and, as a consequence, to the development of chronic liver diseases and to the formation of cancer in this organ [10]. Therefore, hepatic iron homeostasis plays an important role in protecting liver from injuries induced by hepatotoxicants. However, despite evidences reporting the effects of UA on liver toxicity, to these days there is still no data regarding the association of UA on iron metabolism.
In the light of these considerations, the purpose of this study was to investigate the effect of hepatic toxicant UA on the metabolism of iron in rat liver.

Animals, experimental design, and treatments
Male Fischer 344 rats (6 weeks of age) were housed in sterilized cages in a temperature-controlled room (24 °C) with a 12 h light/dark cycle, and given ad libitum access to water and NIH-41 irradiated pelleted diet. After one week of acclimation, the rats were allocated randomly to control and experimental groups. Rats in the experimental group (n = 6 per treatment) were treated by gavage 5 days per week with doses of 60 mg UA/kg bw/day dissolved in DMSO. UA was purchased from Sigma-Aldrich (CAS No. 7562-61-0). Animals in the control groups (n = 6) were gavaged with DMSO only. Body weights were recorded weekly for adjustment of drug dosages. Rats were euthanized by exsanguination following deep isoflurane anesthesia after 6 weeks of treatments. The livers were excised and snap-frozen immediately in liquid nitrogen and stored at -80 °C for subsequent analyses. All experimental procedures were carried out in accordance with the International Rules for the Use of Experimental Animals.

RNA extraction and quantitative reverse transcription-PCR
Total RNA was extracted from liver using miR-Neasy Mini kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA (2 μg) was reverse transcribed using random primers and High Capacity cDNA Reverse Transcription kits (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. cDNA was analyzed in a 96-well plate PCR assay format using a Quant Studio™ 7 Flex Real-Time PCR System (Life Technologies, Carlsbad, CA). Each plate contained experimental genes and a housekeeping gene β-actin; (Actb). All primers for the gene expression analysis were obtained from Life Technologies: transferrin (Tf), transferrin receptor (Tfrc), ferritin light chain (Ftl), ferritin heavy chain 1 (Fth), Solute carrier family 40 member 1 (Slc40a1 (Fpn1)). The relative level of each transcript was determined using the 2 -ΔΔCt method [11].

Analysis of hepatic iron content by inductively coupled plasma mass spectrometry
Inductively coupled plasma mass spectrometry was used to measure the total hepatic iron content in the livers of control and UA-treated rats. Briefly, microwave dissolution of the liver samples was performed with 4.0 ml of concentrated HNO 3 using 100 mg of tissue, followed by quantitative transfer and dilution using 2 % HNO 3 . Tissue microwave dissolution was accomplished by the application of up to 1600 W power, 200 °C for 35 min, utilizing a Microwave-Accelerated Reaction System Model MARS-X (CEM Corporation, Matthews, NC). The iron content was determined with an Agilent 8800 Inductively Coupled Plasma mass spectrometer, utilizing the 56 Fe isotope. 45 Sc, at 100 ng/ml, was used as an internal standard.
Helium collision cell gas was used to eliminate argon oxide ( 40 Ar 16 O + ) poly-atomic interferences.

Statistical analyses
Statistical analyses were performed using Sigma-Plot 13.0 software (Systat Software, Inc, San Jose, CA). Results are presented as mean ± S.D. Data were analyzed by an t-test and one-way analysis of variance (ANOVA), with pair-wise comparisons being made by the Student-Newman-Keuls method. Values of P≤0.05 were considered significant.

Effect of usnic acid on the expression of iron metabolism-related genes in rat liver.
In order to investigate the major mechanisms of impaired iron metabolism during hepatotoxicity, mRNA levels of Tfrc, Tf, Ftl, Fth, and Fpn1 in rat liver that received 60 mg usnic acid/kg bw/day were determined. Fig. 1A shows that the expression of Tfrc decreased by 30 % in liver of rats treated with UA compared with the control. In contrast, the expression level of Ftl and Fth was increased by 16 % and 10 %, respectively, in the rats of the study group.
There were no changes in the level of Tf and Fpn1 in the livers of F344 rats treated with UA. In addition to alterations in the expression of iron-related genes, treatment of rats with 60 mg usnic acid /kg bw/ day resulted in the reduction by 22 % in the hepatic iron content (Fig. 1B).
Discussion. Several studies show toxic impact of UA on liver function both in vitro and in vivo [12,13]. It is well-known that the abnormalities in hepatic iron homeostasis may exacerbate liver injury induced by hepatotoxicants [14]. The results of present study demonstrate that administration of UA led to changes in the expression of iron exchange genes in rat liver, especially an up-regulation of Ftl, Fth genes and a down-regulation of Tfrc.
As mentioned in the introduction, Tfrc gene is known to encode the transferrin receptor 1, which is responsible for iron import from transferrin into cells by endocytosis [15]. For instance, our findings of the decrease in Tfrc in the livers of UA-treated rats correspond to the report by Shpyleva et al. [16] that showed a reduction in Tfrc expression in neoplastically transformed TRL1215 cells, exposed to sodium arsenite.
Ferritin is known to be a major protein that stores excess iron [17], whose expression level was increased in our study. Our previous study of methapyrilene-induced hepatocarcinogenesis demonstrated extensive alterations of Ftl in the livers of F344 rats [18]. In contrast, the level of hepatic iron content was decreased in rats treated with UA. Similar results were demonstrated by Bloomer et al. [19], that a high-fat diet that produced histologic features of nonalcoholic steatohepatitis in mice was accompanied by increased markers of inflammation and oxidative stress and decreased hepatic iron concentrations. Other study has reported that usnic acid had Fe 3+ reducing and Fe 2+ chelating effect [20].
Conclusion. Among the known proteins that regulate iron homeostasis, expression of Tfrc, Ftl and Fthiron metabolism-related genes, have undergone the greatest changes in our study. Thus, the results of this work indicated changes in iron metabolism, specifically, iron reduction due to the action of UA in rat liver.
The authors of this study confirm that the research and publication of the results were not associated with any conflicts regarding commercial or financial relations, relations with organizations and/or individuals who may have been related to the study, and interrelations of coauthors of the article. Стаття надійшла 09.04.2020 р.