Basal Blood Morphology, Serum Biochemistry, and the Liver and Muscle Structure of Weaned Wistar Rats Prenatally Exposed to Fumonisins

Cereals are often contaminated with fumonisins, which are the toxic byproducts of mold. The aim of the study was to determine the effect of maternal exposure to fumonisins on the development and the liver function of the offspring at weaning. Two doses of fumonisins (60 and 90 mg/kg b.w.) were tested. The changes in the basal blood morphology, the biochemical parameters, the absolute and relative weights of the vital organs, and the changes in the cardiac and biceps brachii muscle histology were studied. The liver damage was assessed by evaluating the liver morphology and the common clinical liver panel. Maternal fumonisin intoxication caused a decrease in the body weight at birth and an increase in the heart, liver, kidney, lungs, ovaries, and testes weights. The cytokines and hormones, as well as the red blood cell counts and hemoglobin levels, were elevated in a dose-dependent manner following the exposure to fumonisins. Maternal exposure caused degenerative morphological and structural changes in the liver, as well as inflammation in the striated muscles, such as the heart and biceps brachii, and disproportionate development of the rat offspring in a dose-dependent manner. Moreover, FB exposure resulted in the disproportional development of the rat offspring in a dose-dependent manner, which was probably caused by the bodily hormonal dysregulation. Prenatal fumonisin exposure can be a pathological precursor for serious diseases, such as obesity and diabetes, later in life. The experiment was performed in accordance with the EU Directive 2010/63/EU, under the license of the State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives in Lviv, Ukraine. The FBs were produced in vitro on a maize grain medium with the use of F. moniliforme, as previously described [12]. Briefly, autoclaved, coarsely cracked grains were inoculated with F. moniliforme cultures and were cultivated for 4 weeks at 24 °C. The contaminated maize was then autoclaved, dried, ground, and analyzed for FB1 and FB2 using liquid chromatography, which showed the typical 3:1 ratio of FB1 and FB2 (73% to 27%). Next, the FBs were extracted from the ground grains with an ethanol solution, were quantified using an ELISA (#R3401, Ridascreen Fumonisin, R-Biopharm AG, Darmstadt, Germany), and were concentrated to 100 mg/mL FB1 + FB2 stock solution. During the experiment, the FB extract stock was diluted in 0.9% saline solution to yield the necessary concentration in 0.5 mL on the basis of the daily measurements of individual rat weight. Pregnant Wistar rat dams (n = 18; 5-weeks-old) were housed individually in polypropylene cages (the dimensions of 380 × 200 × 590 mm). During a one-week acclimatization period under laboratory conditions, the dams were kept at a temperature of 21 ± 3 °C and a humidity of 55 ± 5%, with a 12 h/12 h day/night cycle, and were fed a standard laboratory rodents’ diet ad libitum, with free access to water. After the acclimatization period, the rats were randomly allocated to either a control group, which was not treated with FB (C group; n = 6) or to one of the two other groups that were intoxicated with FB at a dose of 60 mg FB/kg b.w. or 90 mg FB/kg b.w. (n = 6 in each group). A standard laboratory rodents’ diet was offered to the pregnant dams, and the animals were fed ad libitum. The fumonisins were given by daily intragastric administration in 0.5 mL of 0.9% saline solution, from the 7th day of pregnancy up to parturition [12,13]. The control animals received saline solution in the corresponding amount and manner. The 90 mg FB/kg b.w. dose was equal to 1/10 of the established LD50 dose [12,18] and was sufficient to induce subclinical intoxication in adolescent rats [12,18]; while the 60 mg dose exceeded the dose required to trigger embryonic neural tube defects when FB were given before the 7th day of pregnancy, was equal to 1/15 of the established LD50 value, and did not induce subclinical or clinical symptoms in adolescent rats [12]. Lower FB doses have been studied extensively (0.1, 0.5, 1.9, 3.8, 6.3, 10, 12, 15, 18, 25, 30, 45, and 50 mg/kg b.w., given in different gestational days) [22]. The higher dose was not used because it results in the presence of clinical signs and for this reason permission from the ethical committee was not received. A trained veterinarian did not note any changes in pregnant dam behavior or basal health state. Following parturition, all newborns were weighed and kept with their mothers, without translocation between the litters. All newborns were divided according to their mothers as follows: the 0 FB group were considered the controls; the 60 FB group were prenatally exposed to FB at a dose of 60 mg/kg b.w.; and the 90 FB were prenatally exposed to FB at a dose of 90 mg/kg b.w. At weaning, at the age of 28 days, four offspring from each mother (two males and two females) (n = 12 males and n = 12 females in total) were weighed and euthanized by CO2 inhalation. After the euthanasia, immediately after the collection of blood samples, the animals were dissected and various organs, including the liver, lungs, heart, testes, ovaries, and kidneys, were removed and weighed individually. The relative organ weight was determined. It is accepted practice in presenting organ weight data to express the results relative to the animal’s body weight. The whole blood was collected by intracardiac puncture into tripotassium salt of ethylenediaminetetraacetic acid (K3EDTA) coated tubes (BD Vacutainer Systems, Plymouth, UK) for hematology and into vacutainer tubes with cloth activator for blood serum determinations (1300× g for 10 min at 18 °C). The collected serum was aliquoted into polypropylene tubes and stored at −86 °C until the assays were performed. The number of white blood cells (WBC), lymphocytes, monocytes, neutrophils, red blood cells (RBC), and platelets (PLT), as well as the hemoglobin concentration (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were determined using an automatic hematology analyzer (Advia 2120, Siemens Healthcare, Erlangen, Germany). The serum was analyzed for the following common markers of liver damage: alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP); and the following other serum parameters: creatine kinase (CK), total bilirubin (TBIL), total cholesterol (TCHOL), glucose, total protein (TP), calcium (Ca), phosphorus (P), magnesium (Mg), and lactate dehydrogenase (LDH), using an automatic analyzer (Mindray BS-120, Bio-Medical Electronics, Shenzhen, China) and ready-to-use commercial tests (Alfa Diagnostics, Warsaw, Poland). All analyses performed were verified with the usage of multiparametric control serum (Alfa Diagnostics, Warsaw, Poland). The serum concentrations of growth hormone (GH), interleukin 1β (IL-1β), and interleukin 6 (IL-6) were determined using commercial, rat-specific, enzyme-linked immunosorbent assay (ELISA) kits from BT-Lab (Korain Biotech, Shanghai, China). The serum insulin (INS) was determined using a commercial, rat-specific, ELISA kit from Qayee-bio (QY-{“type”:”entrez-nucleotide”,”attrs”:{“text”:”E11704″,”term_id”:”22025340″}}E11704; Qayee-bio, Shanghai, China). All procedures were performed in accordance with the manufacturers’ protocols. The analysis of the samples was performed in duplicates, using a microplate spectrophotometer (Benchmark Plus, Bio-Rad Laboratories, Inc., Hercules, CA, USA). To calculate particular results, the individual standard curves that were created in individual tests were used. The samples from the center of the right dorsal lobe of the liver were snap frozen in liquid nitrogen and stored at −80 °C until subsequent analyses. The heart (left ventricle) and a section from the biceps brachii muscles were fixed in 4% buffered formaldehyde (pH 7.0) for 24 h. The formaldehyde-fixed samples were fixed in paraffin according to a routine procedure. The samples were cut with a microtome, depending on the target, into 6-μm (Leica RM2146 microtome, Nußloch, Germany) or 4-µm (Microm HM 360, Microm, Walldorf, Germany) sections and were stained with Goldner trichrome, and hematoxylin & eosin (H&E) (Sigma-Aldrich, Darmstadt, Germany) for morphological examination using light microscopes (CX43 and BX63, Olympus, Tokyo, Japan). The micromorphology of the liver was examined under microscopic observation and the images were collected with the use of graphical analysis software, CellSens Olympus Version 1.5 (OLYMPUS, Tokyo, Japan). A square 9-field grid was used to take consecutive images of each section, and images were taken from the four corner fields and the center field. The microscopic observations identified and evaluated the normal structure of the liver, including the portal triad and terminal hepatic veins, for assessing the architecture of the lobules. Mature fibrous tissue, portal tract stroma, and immature fibrous tissue, as well as lobular architecture and small hepatocytes (as characteristic of regeneration), were also assessed. The microscopic observations also allowed us to identify ballooning degeneration. In addition to the histopathological examination, the total hepatocyte number, the number of binucleated hepatocytes, and the non-hepatocyte cell number were determined on three separate tissue sections, on at least ten different areas of each section, using image analysis software ImageJ 1.53 (National Institute of Health USA, http://rsb.info.nih.gov/ij/index.html; accessed on 24 July 2022). The measurements were then averaged and were expressed as the mean value of the calculated parameters for each rat [23,24]. The histological sections of the left ventricle myocardium and the bicep muscle were analyzed under microscopic observation (Nikon E600, Tokyo, Japan) and inflammatory cell infiltration was assessed semi-quantitatively (0 = lack, 1 = low, 2 = moderate, 3 = high, and 4 = severe). All histological evaluations were performed independently by three histologists with more than 20 years of experience in the field (D.W., E.T., and P.D.). The apoptotic nuclei were detected using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Melbourne, Australia) following the manufacturer’s protocol. Briefly, after deparaffinization and rehydration, slides were pretreated with proteinase K solution (10 μg/mL, Promega Corporation, Madison, WI, USA) for 15 min at room temperature (RT), followed by incubation at RT for 10 min in 3% H2O2 in methanol to quench endogenous peroxidase activity. The slides were then incubated in equilibration buffer for 10 min at RT in a humid chamber, after which the enzyme TdT (terminal deoxynucleotidyl transferase) was added at working strength and they were incubated for 1 h in a humid chamber at 37 °C. The slides were then washed at RT with wash buffer for 10 min, followed by washing with PBS (phosphate-buffered saline) for 5 min. After the excess fluid was drawn off, anti-digoxigenin conjugate was applied directly to the slides and they were incubated for 30 min at RT in a humidified chamber. After washing in PBS, apoptotic cells were visualized by adding DAB (3,3′-diaminobenzidine) solution. A negative control was performed without active TdT enzyme to control for non-specific incorporation of nucleotides or for non-specific binding of enzyme conjugate. All of the sections were counterstained with Mayer’s hematoxylin, dehydrated through an increased series of ethanol, and were mounted under glass with DPX (dibutylphthalate polystyrene xylene, Sigma Chemical Co, St. Louis, MO, USA). The TUNEL-positive nuclei were counted on 10 random areas of each liver tissue and the results were expressed as the percentage of apoptotic cells per 100 randomly counted liver cells. The sections were analyzed under a Zeiss Axio Imager A.2 light microscope (Carl Zeiss AG, Jena, Germany) and AxioVision Digital Image Processing System version 4.8.2 (Carl Zeiss AG, Jena, Germany). The liver tissues for immunohistochemical analysis were frozen in liquid nitrogen and stored at −80 °C for later analysis. The samples were mounted on a cryostat holder using tissue-freezing medium (Tissue-Tek; Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands). The frozen tissue was cut into 10-μm-thick slices at −20 °C in a cryostat (Slee MEV, Mainz, Germany). Beclin-1 activity was determined on frozen sections that were fixed with 4% formaldehyde, as paraformaldehyde (PFA), in 0.1M phosphate buffer (PB) (pH 7.4), and then were incubated for 30 min in 5% normal goat serum (NGS). They were then incubated overnight at 4 °C with anti-Beclin-1 monoclonal antibody (sc-48341, Santa Cruz Biotechnology, Dallas, TX, USA; dilution 1:100).The slides with control for primary antibody were incubated with PBS instead of primary antibody. After several washes in 0.01M sodium phosphate buffer (PBS) containing 0.5% Triton-X, sections were incubated overnight at 4 °C with goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488 (A11001, Thermo Fisher Scientific, Waltham, MA, USA; dilution 1:1000). After final washing, the slides were mounted with Vectashield mounting medium (Vector Labs, Burlingame, CA, USA) with DAPI and were examined with a Zeiss Axio Imager A.2 (Carl Zeiss AG, Jena, Germany) fluorescence microscope and AxioVision Digital Image Processing System version 4.8.2 (Carl Zeiss AG, Jena, Germany). The results are expressed as means ± SEM. At the beginning, the effect of the mother was examined by nested ANOVA, where the offspring were nested into the relevant mothers. Since there was no significant effect of the mothers detected, the rest of the analysis was carried out using a two-way ANOVA, followed by Tukey’s multiple comparison post-test. Planed comparisons were used to check the linear and quadratic effects. The Shapiro–Wilk test and the Brown–Forsythe test were performed to check the assumptions of normal distribution of the data and the equality of the variance, respectively. All statistical analyses were carried out using data analysis software system STATISTICA (ver. 12, StatSoft, Inc., Tulsa, OK, USA). In cases where data lacked normal distribution and/or equality of variance, the non-parametric Kruskal–Wallis and median tests were applied. The following statistical model was used to analyze the selected parameters: where xij—observation (measured parameter), i—the level of the first factor (0 mg FB/kg b.w., 60 mg FB/kg b.w., and 90 mg FB/kg b.w.), j—level of the second factor, sex (female or male), k—number of measurements, µ—constant, general mean, αi—main effect of the first factor, βj—main effect of the second factor, (αβ)ij—interaction effect of the main factors, and εijk—random error. p-values less than 0.05 were considered statistically significant.