Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study

Ethical statement

The study protocol and the procedures for animal care and experiments were approved by the Research Ethics Committee of the Faculty of Dentistry, Ain-Shams University, Egypt (#615).

All efforts were made to ameliorate any suffering of the animals by adopting the OECD 423 test guidelines.

Animals

This study used 21 adult male albino rats, three months old and weighing 200-220 g. The animals were obtained from and housed in the Animal house, Faculty of Medicine, Ain Shams University. Sample size calculation was performed using G*Power version 3.1.9.2 (University Kiel, Germany)¹⁴. The effect size was 0.95 using α level of 0.05 and β level of 0.05, i.e., power = 95%; the estimated sample size (n) was a total of 21 samples for three groups.

Rats were housed separately in cages (Suzhou Suhang Technology Equipment Co., Ltd.) in a constant temperature (22–24°C) and light-controlled room on an alternating 12:12 hour light-dark cycle and had free access to food and water. The animals were fed a natural diet and water ad libitum throughout the whole experiment.

All 21 rats were given a number (1–21) using a marker pen, then randomized by putting the numbers in an envelope and dividing them into three groups according to the numbers which were taken from the envelope

The three groups were as follows :

The rats were injected every morning at 9 am in the animal house laboratory of Ain Shams university.

Rosemary methanolic extract

Dried rosemary leaves were obtained from Harraz for food industry, natural products and botanical herbs (Product number 215). Dried rosemary leaves were ground for methanolic extraction, which was done as described by Hendel et al.¹⁷. The obtained rosemary leaves were powdered and 100 g were submitted to hydrodistillation for 3 h with 1000ml distilled water using Clevenger type apparatus. The extracted oil was collected and dried over anhydrous sodium sulfate, then stored in sealed glass vials in a refrigerator at 4°C until use. From the plant extract material, 30 g was soaked for eight hours in methanol solvent at 40°C in a sterile conical flask. The mixture was then filtered using Whatman No. 4 filter paper to yield a light-brown filtrate. The methanolic filtrate was concentrated using a vacuum rotary evaporator (Heidolph 36001270 Hei-Vap Precision Rotary Evaporator) at 100 rpm for 30 minutes. The remaining extract was then dried in a vacuum oven for two hours to ensure the elimination of any residual solvent. The final extract was a dark green powder, which was then dissolved in 9% saline.

Sample preparation and microscopy

At the end of each experimental period, rats were euthanized by CO2 asphyxiation followed by cervical dislocation and their SMSGs were cut out performed by experienced animal laboratory personnal. SMSGs of the right side were fixed in neutral buffered formalin (10%), embedded in paraffin and sections 6 microns thick were sliced and stained with hematoxylin and eosin (H&E) for light microscopic examination using a Leica DM 1000 light microscope and camera and Leica Application suite-LAS software in the research center, The British University in Egypt. The specimens of the left sides were used for ultrastructural examination where small specimens were cut and fixed in 3% glutaraldehyde and osmium tetraoxide, dehydrated in alcohol and embedded in an epoxy resin. Microtome sections were prepared at approximately 500–1000 um thickness with a Leica Ultracut UCT ultramicrotome. Thin sections were stained with toluidine blue (1X), then sections were examined using a Lica ICC50 HD camera. Ultra-thin sections were prepared at approximately 75–90 um thickness and were stained with uranyl acetate and lead citrate, then examined using a JEOL (JEM-1400 TEM) transmission electron microscope.

Light microscopic results

Histological examination of SMSG of the control group is shown in Figure 1A and Figure 1B, which revealed normal histological features of the submandibular salivary gland.

Figure 1. Photomicrographs of haemotoxylin and eosin (H&E) stained sections in submandibular salivary glands.

(A, B) Control group showing regular gland architecture of serous acini (S) intercalated duct (circle), granular convoluted tubule (GCT), striated duct (ST) and excretory duct (EX) surrounded by fibrous connective tissue stroma (arrows) (H&E orig. mag x 400). (C, D) AFB1 treated group showing remnants of degenerated acini (red arrows), atrophic serous acini (S) with cytoplasmic vacuolation (black arrows), wide interacinar spaces (yellow arrows), vacuolated GCT and striated duct (ST) and excretory duct (EX) with thickened lining epithelium presenting extensive vacuolations (yellow arrows) and some crescent shaped nuclei in its cells (red arrows). Degenerated areas are seen in the surrounding fibrous connective tissue stroma (black arrows) (H&E orig. mag x 400). (E, F) Rosemary treated group showing normal serous acini (S), intercalated ducts (circle), GCT with few cytoplasmic vacuoles (arrows) and striated ducts with normal cell lining (ST). Dilated and congested blood vessels were observed (B) and excretory duct (EX) shows relatively normal lining with slight vacuolations in some areas (arrow) (H&E orig. mag x 400).

Histological sections of SMSG of the AFB1 administered group rats showed acini apparently decreased in size, as revealed by wide interacinar spaces, and numerous different sized cytoplasmic vacuoles were seen in their cells (Figure 1C). Striated ducts showed a decrease in size and loss of basal striations in the lining cells was observed. The presence of abundant cytoplasmic vacuolations were frequently observed in both striated and granular convoluted tubule (GCT) cells. Excretory duct cells showed extensive intracytoplasmic vacuolations and crescent-shaped nuclei. Retained secretory material was frequently found inside its lumen (Figure 1D). Degeneration in the surrounding connective tissue fibers was also noted (Figure 1D).

Histological examination of the SMSGs of the rosemary group rats presented almost identical histological features to the control group except with a few degenerative changes in both the acini and the ducts in some areas where few small intra-cytoplasmic vacuoles were detected. A limited number of congested and dilated blood vessels were observed close to striated ducts (Figure 1D). Some lining cells of the excretory duct exhibited vacuolations in certain areas (Figure 1F).

Electron microscopic results

Control group. Ultrastructural examination of the SMSG of group A rats showed normal serous acini that were spherical in shape containing basally situated rounded nuclei (open-faced nucleus) and a narrow central lumen (Figure 2A). Acinar cells exhibited parallel arrays of rough endoplasmic reticulum (rER) (Figure 2B) and oval-shaped mitochondria (Figure 2C). Secretory granules of variable sizes and different electron densities occupied most of the cytoplasm (Figure 2A). The nuclei of the serous cells appeared euchromatic with a smooth, regular nuclear membrane (Figure 2B). The GCTs presented long columnar cell lining. Their nuclei were large, rounded and basally located. Their cells contained numerous well-circumscribed membrane-bounded electron-dense secretory granules (Figure 2D). The intralobular striated ducts consisted of long columnar cells. The basal cell membrane of the lining cells showed complex extensive infoldings. Few vertically arranged mitochondria were found interspersed with the folded membranes (Figure 2E). The excretory duct was lined by pseudostratified epithelium containing open face nuclei (Figure 2F).

Figure 2.

Electromicrographs of the control group showing (A) acinar cells surrounding acinar lumen (L), rounded basally situated nuclei (N) and many secretory granules (SG) (uranyl acetate and lead x 5000). (B) Part of an acinar open faced nucleus (N) with regular nuclear membrane (arrow) and parallel arrays of rough endoplasmic reticulum (rER) (uranyl acetate and lead x 12000). (C) Normal acinar mitochondria with cristae (uranyl acetate and lead x 12000). (D) Granular convoluted tubule with columnar cells and numerous electron dense secretory granules (SG) and basally situated open faced nucleus (N) (uranyl acetate and lead x 2500). (E) Striated duct cells with open face nucleus (N), numerous basal infoldings (arrows) and radially arranged mitochondria (M) (uranyl acetate and lead x 5000). (F) Excretory duct showing pseudostratified epithelium containing open face nucleus (N) (uranyl acetate and lead x 2500).

AFB1 group. Numerous large vacuolations were observed in the cytoplasm of acinar cells of AFB1 administered rats (Figure 3A). Their nuclei showed irregular outline and clumping of chromatin material and exhibited differences in their shape and size, with variable degrees of shrinkage and pyknosis (Figure 3A, Figure 3B and Figure 3C). rER presented luminal dilatation, discontinuity and fragmentation (Figure 3C). Some acinar cells showed mitochondria with loss of cristae and the presence of vacuoles (Figure 3B). The GCTs showed areas of degeneration in the form of a decrease in the number of electron-dense granules, intracytoplasmic vacuolations and dilatation in the rER (Figure 3D). The irregular shaped cells of the striated duct revealed signs of degeneration displayed as shrunken nuclei and irregular nuclear outline. Degenerated organelles (especially dilated rER) and intra-cytoplasmic vacuolations were frequently encountered in the duct cells. Loss of basal infoldings on the striated ducts was also noticed (Figure 3E). The excretory duct cells showed cytoplasmic vacuolations and nuclei with an irregular outline (Figure 3F).

Figure 3.

Electromicrographs of the AFB1 group showing (A) serous acinus with different sized intracytoplasmic vacuolations (V) and shrunk nuclei (N) (uranyl acetate and lead x 5000). (B) Open face nucleus of serous acinar cell (N) with irregular nuclear membrane (white arrows). Radially arranged mitochondria (M) with dilated cristae and vacuoles. Degenerated cytoplasm (black arrows) can be noticed (uranyl acetate and lead x 12000). (C) Serous cell with irregular nuclear membrane (N) & dilated rough endoplasmic reticulum (rER) (uranyl acetate and lead x 5000). (D) Granular convoluted tubule cells with intracytoplasmic vacuolations (arrows) and dilated rER (uranyl acetate and lead x 4000) (E) Striated duct with indistinct cell outlines and numerous large intracytoplasmic vacuolations (V) pressing on the cell nucleus and loss of basal striations in some cells (uranyl acetate and lead x 5000). (F) Excretory duct cells showing cytoplasmic vacuolations (V) and nuclei (N) with irregular outline (uranyl acetate and lead x 2500).

Rosemary treated group. Electron microscopic examination of SMSG of rats after treatment with rosemary revealed that the majority of acinar cells almost regained their normal architecture (Figure 4A). Slightly dilated rER and very few cytoplasmic vacuolations were observed (Figure 4B). GCTs were lined by long columnar cells with basally situated nuclei and many electron-dense secretory granules (Figure 4C). Striated ducts regained their normal histological appearance, with the columnar cells having numerous basally located radially arranged rod-shaped mitochondria with minimal changes. Desmosomal junctions appeared in a normal pattern in between cells (Figure 4D and Figure 4E). The excretory duct was lined by pseudostratified epithelium containing open face nuclei (Figure 4F).

Figure 4.

Electromicrographs of the rosemary treated group showing (A) serous acinus containing nucleus (N), numerous secretory granules and few cytoplasmic vacuoles (uranyl acetate and lead x 5000). (B) Open face nucleus (N) of an acinar cell and parallel arrays of rough endoplasmic reticulum (rER) with slight dilatations in some areas. Slight perinuclear space can be noticed (arrow) (uranyl acetate and lead x 12000). (C) Granular convoluted tubule (GCT) containing basally situated nuclei (N) and numerous electron dense secretory granules (SG) (uranyl acetate and lead x 2500). (D) Striated duct cell with open face nucleus (N) and tubular shaped mitochondria (M). Minimal cytoplasmic degeneration and some loss of basal infoldings can be observed (black arrows) (uranyl acetate and lead x 12000) (E) Striated duct columnar cells with basal infoldings (black arrows) and numerous rod shaped mitochondria (white arrows) (uranyl acetate and lead x 5000). (F) Excretory duct with pseudostratified epithelium lining cells presenting open face nucleus (N) (uranyl acetate and lead x 2500).

Original microscopy light and electron microscopy images for all groups are provided as Underlying data¹⁸.

Underlying data

Figshare: Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study. https://doi.org/10.6084/m9.figshare.12644855.v2¹⁸

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).