Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers.
The physiological role of the vitamin biotin (also named B7) is to participate as a coenzyme of carboxylases [
Pharmacological doses of biotin have hypolipidemic [
In previous studies in our laboratory, normal mice fed a biotin-supplemented diet for 8 weeks demonstrated increased insulin secretion and higher levels of mRNAs involved in the control of insulin transcription and secretion compared with mice fed a biotin-sufficient diet [
Other studies have shown that pharmacological doses of biotin modify tissue morphology. Our research group previously demonstrated that treatment with pharmacological concentrations of biotin decreases the number of primary and Graafian follicles in female mice [
Because of biotin’s effects on glucose and lipid metabolism, supplements and medications containing pharmacological amounts of the vitamin are commercially available [
All interventions were approved by the Ethical Committee for Experimentation of the Biomedical Research Institute of the National Autonomous University of Mexico. At weaning three-week-old male Balb/cAnN mice were fed one of the following diets: biotin-control (TD-01362) or biotin-supplemented diet (TD-02458) containing 1.76 mg and 97.70 mg of free biotin/kg diet, respectively (Harlan Teklad, Madison WI, USA) as described previously [
Blood samples were collected and centrifuged at 1600 ×g and 4°C for 10 min. Sera were stored at −20°C until used. Hemolyzed samples were discarded to avoid interference with the assays. The compounds were quantified by colorimetric assays (Spinreact, Sant Esteve de Bas, Spain) according to the manufacturer’s protocol and were expressed as U/L.
Lipid peroxidation was quantified in liver homogenates by assaying malondialdehyde using the thiobarbituric acid by method [
Immediately after the mice were sacrificed, liver samples were washed in a saline isotonic solution to eliminate remnant blood, homogenized in 5% ice-cold metaphosphoric acid (1/20 w/v) (Sigma Aldrich, St. Louis, MO, USA), and centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was collected and kept on ice. Glutathione was quantified spectrophotometrically at 405 nm with the HT Glutathione Assay Kit (Trevigen, St Louis, MO, USA) according to the manufacturer’s recommendations. The concentration of reduced gluthatione (GSH) was calculated using a GSH standard curve. Oxidized glutathione contents were determined by subtracting the amount of GSH from the level of total glutathione.
Approximately 100 mg of liver was homogenized with a glass potter homogenizer in an ice-cold saline isotonic solution. Tissue extracts were centrifuged at 12500 ×g for 30 min at 4°C to remove insoluble material, and the protein concentration was measured using a Bio-Rad protein assay following the manufacturer’s instructions. Super oxide dismutase activity was quantified in liver homogenates with a commercial kit based on xanthine oxidase and a color agent according to the protocol provided by the manufacturer (Sigma Aldrich, San Louis, MO, USA).
Catalase activity was measured according to Góth, 1991 [
The livers were dissected and fixed in 10% neutral formalin, dehydrated in ascending grades of alcohol, and embedded in paraffin wax. Consecutive 5
Two slides were prepared for each mouse. These contained three sections of each liver. Ten field areas for each section were randomly selected and examined for histological changes (25x) under light microscope. The random field areas were scored as follows: normal appearance (—), minimal cellular disruption in less than 1% of field area, mild cellular disruption of 1%–30% of field area, moderate cellular disruption of 31%–60% of field area, severe cell disruption of 61%–90% of field area, and very severe cellular disruption of 91%–100% of field area [
To assess binucleated hepatocytes and nucleomegaly, three field areas of six sections were taken from 9 mice in each group. These were analyzed at 40x in an Olympus BX51 microscope. The analyses were performed simultaneously by two persons who were blinded to the group identity.
Liver homogenates were prepared as described above and were treated with proteinase K [10 mg/ml] to remove interfering proteins. Quantification was performed with a commercial kit
Statistical analyses were performed using the Statview statistical analysis program and GraphPad Prism 6.0 software (Berkeley, CA, USA). All data are presented as the mean ± SEM;
We studied how biotin supplementation influenced liver enzyme tests (Table
Effect of eight weeks of biotin supplementation on serum liver damage indicators.
Enzyme/metabolite | Units | Control | Biotin-supplemented |
---|---|---|---|
Aspartate aminotransferase | [U/L] | 163 ± 11.0 | 196 ± 8.60 |
Alanine aminotransferase | [U/L] | 32.9 ± 3.29 | 30.2 ± 2.40 |
Gamma-glutamyltransferase | [U/L] | 2.77 ± 0.38 | 2.48 ± 0.50 |
Alkaline phosphatase | [U/L] | 164 ± 11.0 | 196 ± 8.63 |
Total bilirubin | [ |
0.30 ± 0.040 | 0.22 ± 0.029 |
Indirect bilirubin | [mg/dL] | 0.29 ± 0.071 | 0.22 ± 0.020 |
Albumin | [mg/dL] | 3.28 ± 0.26 | 2.57 ± 0.24 |
Urea | 52.3 ± 1.21 | 50.7 ± 1.89 |
Values are mean ± SEM.
We also evaluated the effects of biotin supplementation on lipid peroxidation and oxidized and reduced glutathione. Eight weeks of biotin supplementation did not modify malondialdehyde concentrations versus the control group (Figure
Effect of biotin supplementation on liver malondialdehyde and reduced and oxidized glutathione in mice fed a biotin-supplemented diet for 8 weeks. (a) Malondialdehyde concentrations. Values represent the mean ± SEM of 5 mice from each group. (b) Reduced and oxidized glutathione. Black bars: control group; patterned bars: biotin-supplemented group. Values represent the mean ± SEM of 8 mice from each group. All measurements were performed in triplicate.
Glutathione is a major antioxidant that protects tissues from free radical injury. The analysis of glutathione concentrations (Figure
Liver superoxide dismutase activity (Figure
Effect of biotin supplementation on superoxide dismutase and catalase activity in mice fed a biotin-supplemented diet for 8 weeks. (a) Liver superoxide dismutase activity. Values represent the mean ± SEM of 5 mice from each group. (b) Liver catalase activity. Black bars: control group; patterned bars: biotin-supplemented group. Values represent the mean ± SEM of 9 mice from each group. All measurements were performed in triplicate.
Despite the fact that biotin supplementation had no effect on liver toxicity markers, histological analysis showed noticeable differences between groups. Light microscopy observations of livers from control mice showed a normal morphology with adjacent normal sized sinusoids radiating from the central veins toward the periphery of the liver lobules and a normal portal triad (Figure
Effect of biotin supplementation on liver morphology in mice fed a biotin-supplemented diet for 8 weeks. Paraffin sections stained by hematoxylin and eosin. (a) Control group’s representative image showing a normal portal triad. (b) Supplemented group. Left panel: representative image, showing an altered triad with dilated sinusoids and increased number of bile ducts. Right panel: enlarged portal vein with adjacent portal vein branch. pv: portal vein; ha: hepatic artery; bd: bile duct; ds: dilated sinusoids; pvb: portal vein branch. Scale bar represents 100
Changes were also observed in the hepatocyte nuclei. Compared to the mice fed a control diet, the biotin-supplemented group showed a 39% increase in the number of binucleated hepatocytes (control = 13.2 ± 1.29; supplemented = 18.7 ± 1.50%) (Figures
Effect of biotin supplementation on binucleated hepatocytes and nucleomegaly in mice fed a biotin-supplemented diet for 8 weeks. (a) Hematoxylin and eosin representative image of hepatocytes from control and biotin-supplemented group (right) showing mono and binucleated hepatocytes. (b) Quantification of binucleated hepatocytes per field. (c) Hematoxylin and eosin images of hepatocytes from control and biotin-supplemented group (right) showing nuclei <12 and >12
The increased vascularization observed in histology prompted us to determine nitric oxide concentrations because nitric oxide has been proposed to participate in the biotin transduction signaling pathway cGMP/PKG [
Effect of biotin supplementation on nitric oxide concentrations in mice fed a biotin-supplemented diet for 8 weeks. Values represent the mean ± SEM of 9 mice from each group. Measurements were performed in triplicate. Black bars: control group; patterned bars: biotin-supplemented group.
In this study, we found that eight weeks of dietary biotin supplementation induced noticeable changes in liver morphology in normal mice and that these modifications were not paralleled with liver damage markers. The biotin-supplemented mice had a higher percentage of dilated sinusoids, vascularity, and bile ducts compared with the controls. We also found an increased number of binucleated hepatocytes (about 39%) and hepatocytes with nucleomegaly (66%) in the biotin-supplemented mice compared with the controls. These results together with previous results in pancreatic islets [
Binucleated hepatocytes and nucleomegaly are the result of polyploidy [
In previous studies, we observed that normal mice fed a biotin-supplemented diet (approximately 13.5 mg/kg body weight) showed increased pancreatic islet size as well as increased alpha-cells at the islet center compared with the controls [
Changes on ovary histology were also observed by Paul et al. [
Other investigations found that biotin supplementation modified the histopathological features produced in the diabetic state [
Despite the effects of biotin on liver morphology, our data did not find changes on liver damage indicators such as albumin, total and direct bilirubin, gamma-glutamyltransferase, alanine aminotransferase, alkaline phosphatase, or urea. These results agree with studies in normal rats showing that diets containing up to 1,000 mg/kg diet did not affect these parameters [
The reversion of altered tissue structure induced by biotin in diabetic animal models is associated with reduced oxidative stress [
Nitric oxide has an important role in vasodilatation [
The mechanisms involved in biotin-induced histological changes are largely unknown. In pancreatic islets, we found that the changes produced by biotin supplementation on islet architecture might be related to diminished expression of the neural cell adhesion protein
Biotin products with pharmacological concentrations of biotin are commercially available. Biotin administration is considered harmless in humans and rodents [
Our data shows for the first time that biotin supplementation affects liver morphology in normal mice and that these modifications are not paralleled with changes in classical liver damage indicators and oxidative stress markers. These results indicate that biotin toxicity studies need to be addressed with different tools because the pharmacological concentrations of biotin affect tissue morphology and have nuclei effects.
The authors declare that there are no competing interests regarding the publication of this paper.
Dr. Rodolfo Rodríguez-Jurado, Dr. Rosa María Vigueras-Villaseñor of Laboratorio Biología de la Reproducción, and M en C Gerardo Barragán-Mejía of Laboratorio Neurociencias acknowledge Instituto Nacional de Pediatría Departamento de Patología, for histology facilities. The authors are grateful to Dr. Maria Eugenia Gonsebatt Bonaparte, Dr. José Pedraza Chaverri, and Dr. Javier Espinosa Aguirre for valuable discussions throughout these studies, and to M. en C. Betzabé Linares Ferrer for computer assistance. This work is supported by research grant from the Consejo Nacional de Ciencia y Tecnología, 219787-M and 99294-M, and Fondos Federales 074/2013. M. en C. Leticia Riverón-Negrete is a doctoral student from the Doctorado en Ciencias Biológicas at Universidad Nacional Autónoma de México (UNAM). Jonathan Alcántar-Fernández is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, and was the recipient of Fellowship 384151 from Consejo Nacional de Ciencia y Tecnología (CONACYT), México.