Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

Study of zeolite clinoptilolite d-glucose adsorption properties in vitro and in vivo.

Beneficial effects of a natural zeolite clinoptilolite in vivo on mammals, including humans, have been empirically observed and documented in literature. The positive biological activities have been associated to its detoxifying and antioxidative properties, and its immunostimulative and adsorption properties. Herein, we present the in vitro and in vivo study of clinoptilolite zeolite materials adsorption properties for d-glucose. In particular, we present data on the interaction of d-glucose on the tested zeolites' surface obtained by scanning electron microscope (SEM) and Energy-dispersive X-ray spectroscopy (EDS) and quantification by ultra high-performance liquid chromatography (UHPLC). We also present results on the reduction of blood glucose levels in mice pre-treated with clinoptilolite in vivo upon feeding with d-glucose. In vivo results were in line with the in vitro adsorption and/or interaction properties of tested zeolite materials for d-glucose and were quantified by UHPLC as well (11.34% for TMAZ; 10.82% for PMA and 8.76% for PMAO2). In vivo experiments in mice showed that PMA zeolite reduces blood glucose levels upon 15 min for 13% (at p < 0.05) up to 19.11% upon 120 min (without statistical significance) in clinoptilolite pre-treated mice fed by addition of d-glucose. Due to lack of explicit mechanistic knowledge on zeolite clinoptilolite interactions or adsorption with sugars in vitro and in vivo, presented study provides novel insights into these aspects for researchers in the field. The presented data merit further investigations as the material clearly shows a potential in management of hyperglycemia, such as for example in obese people, people with diabetes and people with metabolic syndrome where it could help regulate blood glucose levels.

Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents.

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest peptidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd.

Design, Synthesis, and Biological Evaluation of Desmuramyl Dipeptides Modified by Adamantyl-1,2,3-triazole.

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment able to trigger the immune response. Structural modification of MDP can lead to the preparation of analogs with improved immunostimulant properties, including desmuramyl peptides (DMPs). The aim of this work was to prepare the desmuramyl peptide (L-Ala-D-Glu)-containing adamantyl-triazole moiety and its mannosylated derivative in order to study their immunomodulatory activities in vivo. The adjuvant activity of the prepared compounds was evaluated in a murine model using ovalbumin as an antigen, and compared to the reference adjuvant ManAdDMP. The results showed that the introduction of the lipophilic adamantyl-triazole moiety at the C-terminus of L-Ala-D-Glu contributes to the immunostimulant activity of DMP, and that mannosylation of DMP modified with adamantyl-triazole causes the amplification of its immunostimulant activity.

Metabolic Disturbances in Rat Sublines with Constitutionally Altered Serotonin Homeostasis.

Central and peripheral serotonin (5HT) have opposing functions in the regulation of energy homeostasis. Both increasing 5HT signaling in the brain and decreasing 5HT signaling in the periphery have been proposed as potential treatments for obesity. This study investigates the relationship between constitutionally high or low 5HT activity and systemic net energy balance. Two sublines of rats with high and low whole-body 5HT tone, obtained by selective breeding for platelet 5HT parameters, were examined for fat accumulation in different white adipose tissue (WAT) depots, glucose/insulin tolerance, blood metabolic parameters, and expression of various metabolic genes. High-5HT animals, unlike their low-5HT counterparts, developed widespread intra-abdominal obesity associated with glucose and insulin intolerance, which worsened with age. They also had elevated blood glucose and lipid parameters but showed no significant changes in circulating leptin, resistin, and adipsin levels. Surprisingly, adiponectin levels were increased in plasma but reduced in the WAT of high-5HT rats. A limited number of metabolic genes belonging to different functional classes showed differential expression in WAT of high-5HT compared to low-5HT rats. Overall, a constitutive increase in 5HT tone is associated with a positive energy balance acting through subtle dysregulation of a broad spectrum of metabolic pathways.

Design, synthesis and biological evaluation of immunostimulating mannosylated desmuramyl peptides.

Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adamantane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest enhancement of IgG production was stimulated by compound 21 (Man-OCH2-ᴅ-(1-Ad)Gly-ʟ-Ala-ᴅ-isoGln).

MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS.

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells.

The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

INTRODUCTION: The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. OBJECTIVES: Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). METHODOLOGY: The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. RESULTS: Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. CONCLUSIONS: Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

Micronucleus, cell-free DNA, and plasma glycan composition in the newborns of healthy and diabetic mothers.

Diabetes is associated with certain environmental exposures, heritable factors, and metabolic conditions of intrauterine development due to diabetes in the mother. We evaluated genomic damage, cell-free DNA, N-glycosylation of umbilical cord plasma proteins (PG), and nuclear division index (NDI) as possible prognostic biomarkers of health risk in the newborns of mothers with treated pregestational diabetes (NBDM; 22 mothers), compared these parameters with those from newborns of healthy mothers (NBHM; 89 mothers), and associated the results with the mothers' lifestyle in both groups, based on a detailed questionnaire. Genomic damage was estimated by the in vitro micronucleus (MN) assay. NDI was detected on MN slides. Glycans were analyzed by ultra-performance liquid chromatography that separates the plasma N-glycome into 46 glycan peaks. Cell-free DNA was analyzed by real-time PCR. For the association between biomarkers and individual characteristics, generalized linear/nonlinear analysis was performed. No significant difference was found between NBHM and NBDM for cell-free DNA levels. There was no association between cell-free DNA levels and lifestyle. MN frequency was significantly higher in NBDM than in NBHM (median, 0.6 vs. 0.3%, p<0.001). MN frequency and NDI were significantly associated with residence (urban vs. rural). PG differed significantly between NBHM and NBDM (p<0.001). A significant association was found between PG and increase of MN frequency (p<0.001). As both MN frequency and altered N-glycosylation are associated with cancer risk, our study indicates need for further investigations.

Gender Difference in Distribution of Estrogen and Androgen Receptors in Intestinal-type Gastric Cancer.

BACKGROUND: Gender difference in survival of patients with gastric cancer is not well investigated. The aim of this study was to analyze the gender-related distribution of estrogen receptor alpha (ERα) and androgen receptor (AR) in the epithelium and stroma of intestinal-type gastric cancer. MATERIALS AND METHODS: Immunohistochemical analysis was performed in 60 patients (42% females). RESULTS: In gastric cancer patients, frequency of ERα-positive cells was lower in epithelium than in healthy individuals, but not significantly. In stroma and epithelium, AR-positive cells were absent from samples of women with T1 and T2 stage disease, while in men, their frequency was significantly increased in stroma of those with T3 and T4 stages and was significantly higher compared to women. AR-positive cells in stroma were fibroblasts, myofibroblasts and mast cells. CONCLUSION: To our knowledge, this study is the first to show gender differences in the distribution and frequency of AR-positive cells in neoplastic stroma of gastric cancer.

Age and sex differences in genome damage between prepubertal and adult mice after exposure to ionising radiation.

The mechanisms that lead to sex and age differences in biological responses to exposure to ionising radiation and related health risks have still not been investigated to a satisfactory extent. The significance of sex hormones in the aetiology of radiogenic cancer types requires a better understanding of the mechanisms involved, especially during organism development. The aim of this study was to show age and sex differences in genome damage between prepubertal and adult mice after single exposure to gamma radiation. Genome damage was measured 24 h, 48 h, and 72 h after exposure of 3-week and 12-week old BALB/CJ mice to 8 Gy of gamma radiation using an in vivo micronucleus assay. There was a significantly higher genome damage in prepubertal than in adult animals of both sexes for all sampling times. Irradiation caused a higher frequency of micronuclei in males of both age groups. Our study confirms sex differences in the susceptibility to effects of ionising radiation in mice and is the first to show that such a difference occurs already at prepubertal age.

Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

The effect of Ca2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca2+ ions (0.42 mM), K2[B3O3F4OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca2+ ions (9.42-72.42 mM), at the same concentration of K2[B3O3F4OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K2[B3O3F4OH], in the medium with a low concentration of Ca2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Testosterone-induced micronuclei and increased nuclear division rate in L929 cell line expressing the androgen receptor.

Data on the possible genotoxic effects of testosterone are limited: in particular, the potential clastogenic and/or aneugenic effects have not yet been properly investigated. An in vitro micronucleus (MN) assay was performed on L929 cells exposed to testosterone at doses of 10, 15, 20, 30, and 40 µg/mL. Significantly increased MN frequencies were detected at doses of 20, 30, and 40 µg/mL after 24 h and 48 h of incubation. The nuclear division index was higher after 48 h than 24 h of incubation. A benchmark dose (BMD) calculation was used to estimate the 1% extra risk level for MN and increased tetranucleated cells. The calculated 1% extra risk level for MN at 24 h was 12.01 µg/mL, with a 95% lower confidence limit (BMDL) at 8.98 µg/mL; the corresponding BMD and BMDL at 48 h were 17.35 µg/mL and 10.69 µg/mL, respectively. The BMD for tetranucleated cells at 24 h was 14.86 µg/mL, with a BMDL of 7.75 µg/mL; the corresponding values at 48 h were 0.50 µg/mL for BMD and 0.87µg/mL for BMD. These findings suggest that the intensity of the mitogenic effect of testosterone increases upon prolonged exposure. The results of our study show that testosterone acts both as a mitogenic and genotoxic agent in L929 cells.

Modulation of γ2-MSH hepatoprotection by antisense peptides and melanocortin subtype 3 and 4 receptor antagonists.

Melanocortins, i.e., melanocyte stimulating hormones (MSH) are peptides with strong antiinflammatory effects. The most investigated aspects of γ2-MSH are related to cardiovascular effects and natriuresis, with limited research available about its anti-inflammatory and cytoprotective effects. The aims of this study were: 1) to examine the effects of γ2-MSH and its derivative [D-Trp(8)]-γ2-MSH on the acetaminophen model of liver damage in CBA mice; 2) to evaluate the modulation of γ2-MSH hepatoprotection by melanocortin subtypes 3 and 4 receptor antagonists SHU 9119 and HS 024; 3) to define the importance of central MSH pharmacophore region (HFRW) by using antisense peptides LVKAT and VKAT. In this study, specific antagonists and antisense peptides were used to target central pharmacophore region of γ2-MSH and [D-Trp(8)]-γ2-MSH, enabling the evaluation of hepatoprotection from the standpoint of the receptor and pharmacophore blockade. The criteria for monitoring the effects of the hormones on the liver damage were alanine transaminase, aspartate transaminase activities (U/L), and pathohistological scoring of liver necrosis (scale 0-5). γ2-MSH (0.24 mg/kg) indicated hepatoprotective effects in comparison to control (p < 0.001). In contrast, [D-Trp(8)]-γ2-MSH did not show any hepatoprotective effects. Application of antagonists SHU 9119 and HS 024, and antisense peptides LVKAT and VKAT, also did not show any hepatoprotective effects. In fact, when combined with γ2-MSH, it annulled its hepatoprotective effect. The results provide evidence for hepatoprotective and antiinflammatory effects of the γ2-MSH in the liver.

In vitro and in vivo antitumor activity of the halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]).

Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for cancer diseases. For in vitro and in vivo investigation of its antitumor effects 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. The detailed in vitro investigation undoubtedly showed that K2[B3O3F4OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration so that aqueous solution of K2[B3O3F4OH], the concentrations of 10(-4) M and less, does not affect cell growth, but the concentrations of 10(-3) M or more, significantly slows cells growth. B16F10 and SCCVII cells show higher sensitivity to the cytotoxic effects of K2[B3O3F4OH] compared to 4T1 cells. Under in vivo conditions, K2[B3O3F4OH] slows the growth of all three tumors tested compared to the control, and the inhibitory effect was most pronounced during the application of the substance. There is almost no difference if K2[B3O3F4OH] was applied intraperitoneally, intratumor, peroral or as ointment. Addition of 5-FU did not further increase the antitumor efficacy of K2[B3O3F4OH].

Interactions with polynucleotides and antitumor activity of amidino and imidazolinyl substituted 2-phenylbenzothiazole mesylates.

Based on previously reported antiproliferative activity screening, four most promising disubstituted 2-phenylbenzothiazole hydrochlorides were chosen for detailed study. Water solubility, as well as liphophilicity/hydrophilicity balance of organic core were modified by conversion to mesylate salts. For purpose of structure/activity studies their structures were determined by X-ray structure analysis. Detailed analysis of interactions of new compounds with double stranded (ds-) DNA/RNA by UV/Vis and CD titrations, thermal melting and viscometry experiments revealed that most of studied compounds intercalate into ds-RNA but bind into minor groove of AT-DNA, and agglomerate along GC-DNA. Furthermore, compounds also interact with ss-RNA, but only amino-imidazolinyl 2-phenylbenzothiazole, 4b displayed well defined orientation and dominant binding mode (by induced CD signals) with poly A and poly G. Besides, in vitro investigations revealed moderate to high antiproliferative activity of benzothiazoles against seven human cancer cell lines, while in some cases (HTC 116, SW620, MIA PaCa-2) high correlation between the type of the amidino group and cytotoxic activity was observed.

Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice.

Recent histopathological investigations in patients with hepatitis suggested possible involvement of Met-enkephalin and its receptors in the pathophysiology of hepatitis. Consequently, we evaluated the potential hepatoprotective effects of this endogenous opioid pentapeptide in the experimental model of acetaminophen induced hepatotoxicity in male CBA mice. Met-enkephalin exhibited strong hepatoprotective effects in a dose of 7.5 mg/kg, which corresponds to the protective dose reported for several different animal disease models. In this group plasma alanine aminotransferase and aspartate aminotransferase enzyme activities, as well as liver necrosis score were significantly reduced in comparison to control animals treated with physiological saline (p>0.01). The specificity of the peptide hepatoprotection was investigated from the standpoint of the receptor and peptide blockade. It was concluded that Met-enkephalin effects on the liver were mediated via δ and ζ opioid receptors. Genotoxic testing of Met-enkephalin confirmed the safety of the peptide.

Comparison of clenbuterol and salbutamol accumulation in the liver of two different mouse strains.

In the European Union, ß(2)-adrenergic agonists like clenbuterol and salbutamol are banned from use as growth promoters. Although clenbuterol and salbutamol both accumulate in the liver, differences in the accumulation rate can be seen among animal species due to different ß(2)-adrenoreceptor distributions. The aim of this study was to compare the accumulation of the two in the liver tissue of two different mouse strains. The study included 200 8-week-old BALB/c and C57/BL/6 mice. One group of BALB/c (40) and one group of C57/BL/6 (40) mice were treated with 2.5 mg/kg body mass clenbuterol per os for 28 days. The remaining two animal groups were treated with salbutamol in the same manner. The animals were then randomly sacrificed on day 1, 15 and 30 post treatments. Despite of the same treatment dose, the results revealed clenbuterol to persist in the liver tissue longer than salbutamol. On post treatment day 30, the concentration of clenbuterol residue in C57/BL/6 and BALB/c mice liver tissue were 0.23 ± 0.02 and 0.21 ± 0.03 ng/g, respectively, while residues of salbutamol were not detected. When comparing the accumulation of both compounds between the two mouse strains, it becomes apparent that no significant difference (P > 0.05) in the accumulation rate can be found.