Effect of honey on mycotoxin production
The in vitro investigations have shown that the biomass of A. parasiticus was enhanced in the medium containing 32% honey. However, biomas of A. ochraceus was decreased in medium containing 32 and 48% honey. Ochratoxin A was not produced at either honey concentrations. On the other side, aflatoxins B1, B2, G1 and G2 production was increased in medium containing 32% honey but decreased in medium of high honey concentration with no production of aflatoxin B2 and G2 (Table ). Generally we did not find any positive relationship between mycelium weight and mycotoxins production.
Effect of honey on mycelium mass and mycotoxins concentration.
Effect of honey on colonic glucuronidases activity
The effects of endogenous colonic probiotic bacteria on bacterial β-glucuronidases activity in the caecum content are shown in table . The level of glucuronidases was very low and no significant differences were observed in its in caecum content between all groups (P < 0.05).
Effect of various mycotoxin and honey treatments on bacterial glucuronidases activity in the caecum content of mice.
Genotoxic effects of mycotoxins on bone marrow cells
The present study revealed that the oral administration of aflatoxins (B1, B2, G1 & G2) and ochratoxin A to male mice induced structural and numerical chromosomal aberrations (Table & Figure ).
Mean values of different types of chromosomal aberrations in bone marrow cells of male mice treated with ochratoxin A and aflatoxins (B1, B2, G1 and G2).
Figure 1 Metaphase spreads of male mice treated with ochratoxin A and aflatoxins showing: a) centromeric attenuation. b) deletion. C) chromatid break (small arrow), fragment (large arrow) of bone marrow cells and d) polyploidy. e) autosomal univalent. F) x-y univalent, (more ...)
Structural chromosomal aberrations were recorded as chromatid gaps, chromatid breaks (Figure ), centromeric attenuations (Figure ), deletions (Figure ) and fragments (Figure ). Table , represents the mean values of different structural chromosomal aberrations induced by mycotoxins in bone marrow cells of male mice. The results show low frequency in chromatid gaps, breaks and fragments, whereas the frequency of centromeric attenuation were significantly high between AT group and HAT group from one side and between AT group and the control groups (NTC and HTC) from the other side.
A significant difference (P < 0.05) was also found in deletions (Figure ) between OT group and HOT group, also between OT group and the control groups (NTC and HTC), which can be attributed to the protective effect of honey.
Significant increase (P < 0.05) in the frequency of total structural chromosomal aberrations was found between OT group and HOT group from one side and between group OT and the control groups (NTC and HTC) from another side. Also, a significant difference was found between AT group comparing to HAT group and the control groups (NTC and HTC), respectively.
Genotoxic effects of mycotoxins on germ cells (spermatocytes)
The results of our study revealed that oral treatment with ochratoxin A and aflatoxins (B1, B2, G1 & G2) induced numerical and structural chromosomal aberrations in germ cells of male mice (Table ). Numerical aberrations were recorded as peridiploidy (n ± 1 or n ± 2) and polyploidy (Figure ). Peridiploidy observed in spermatocytes of male mouse was clearly different (p ≤ 0.5) only in the aflatoxins treated group (AT) compared to the control groups (NTC and HTC). However, polyploidy induced by ochratoxin A treated group (OT) differed significantly (P < 0.05) when compared to the control groups. Meanwhile, the total numerical aberrations were significantly different (P < 0.05) in both ochratoxin A (OT group) and aflatoxins treated group (AT) in comparison to the control groups (NTC and HTC). The protective effect of honey was apparent only in HAT group.
Mean values of different types of chromosomal aberrations in spermatocyte cells of male mice treated with ochratoxin A and aflatoxins (B1, B2, G1 and G2).
Treatment with ochratoxin A highly induced structural chromosomal aberrations especially x-y univalents (Figure ), where significant difference (P < 0.05) was found between OT and HOT groups, also between OT and the control groups (HTC).
Effect of honey on cell proliferation
Microscopic examination of the control liver tissue showed hexagonal hepatic lobules consisting of central vein from which hepatic cords, sinusoids lined by radiating kupffer cells as well as bile canaliculi were apparent. The portal tract was located between three hepatic lobules and consisted of portal vein, hepatic artery and bile duct (Figure ).
Liver of NTC group. Normal liver tissue (H x. & E. ×100).
Administration of ochratoxin A affected the liver tissue as it induced dilated congested central veins, with marked sinusoidal dilation, congestion and hyperplastic kupffer cells (Figure ). Also focal hepatic necrosis, predominantly perivascular was evident (Figure ). Administration of honey with ochratoxin A revealed no beneficial effect as the liver in AOT group was affected the same as in group OT. On the other hand, administration of aflatoxins caused cloudy swelling in hepatocytes, extensive variable side necrotic areas affecting the hepatic parenchyma and infiltrated by neutrophils and macrophages (Figure ). Also, congested dilated central veins and marked sinusoidal dilatation with hupffer cell hyperplasia were also evident (Figure ). The portal tracts are thickened by mononuclear inflammatory cells formed mainly of lymphocytes and telangiectatic, congested vascular channels (Figure ).
Liver of OT group. Focal hepatic necrosis predominantly peri vascular (Hx. & E. ×200).
Liver of OT group. Dilated congested central veins marked sinusordal dilatation & congestion (Hx. & E. ×200).
Liver of AT group. Variable-sized necrotic areas of the hepatic parenchyma infiltrated by neutrophils.(Hx. & E. ×100).
Liver of AT group. Congested dilated centeral veins and marked sinusoidal dilatation (Hx. & E. ×400).
Liver of AT group. Portal tracts thickened by marked dilatation & congestion of vascular channels and mild mano-nuclear inflammatory infiltrate (Hx. & E. ×400).
Histopathlogical examination of the control kidney tissues showed glomeruli, proximal and distal convoluted renal tubules, the medulla contained the collecting tubules and parts of the ascending and dexcending loop of henle. Ochratoxin A administration induced cloudy swelling of proximal convoluted tubules, fibrin thrombin within glomerular capillary loops, increased number and size of glomeruli (Figure ) and interstitial fibrosis and congestion (Figure ). On the other hand, co-administration of honey with ochratoxin A ameliorated the effect of ochratoxin A on the kidney where vascular congestion returned to normal. However, they were still slightly enlarged (Figure ). Administration of aflatoxins induced cloudy swelling of the epithelial lining of renal tubules with mild interstitial fibrosis and congestion (Figure ).
Kidney of OT group. Increased number & size of glomeruli (Hx. & E. ×100).
Kidney of OT group. Interstitial fibrosis and congestion (Massons trichrome ×100).
Kidney of HOT group. Return to normal except for vascular congestion (Hx. & E. ×200).
Kidney of AT group. Cloudy swelling of epithelium of tubules (Hx. & E. ×100).
Regarding the effect of ochratoxin administration on brain, histopathlogical examination pointed to the presence of focal areas of degeneration (Figure ), while Co-administration of honey with ochratoxin A ameliorated this effect. In addition, administration of aflatoxin did not induce any changes in brain tissue.
Brain. Focal areas of degeneration in the OT group (Hx. & E. ×100).
Histopathlogical examination of the control lung showed thin alveolar walls, consisted of epithelial cells in both sides of centrally, located capillaries with no intervening connective tissue stroma. Administration of ochratoxin A affected the lung tissues where, the lung showed edema, congestion, lymphoid aggregates in interstitial tissue (Figure ). The results pointed out that the lung showed relief of congestion (co-administration of honey with ochratoxin A) (Figure ). Whereas, the lungs showed interstitial fibrosis and inflammatory infiltrate after administration of aflatoxin (Figure ).
Lung of OT group. Edema, congestion, lymphoid aggregates of interstitial tissue (Hx. & E. ×100).
Lung of HOT group. Relief of congestion but edeme & lymphocytic aggregates are still present (Hx. & E. ×100).
Lung of AT group. Showing interstitial fibrosis, inflammatory infillate with focal lymphocytic aggregates (Massons trichrome ×100).
The control spleen tissue showed connective tissue framework (which includes capsule, trabeculae and reticular connective tissue) and a parenchyma of lymphoid tissue in the form of white (lymphoid nodules) and red pulp (Figure ). Figure illustrated the effect of ochratoxin A on the spleen where it showed expansion of the white pulp with destruction of normal architecture, adventitial thickening of arterioles, adventitial thickening of arterioles focal fibrosis of the red pulp and compression of the sinusoids.
Spleen of NTC group. Induced similar histopathlogical, white & red pulps. (Hx. & E. ×100).
Spleen of OT group. Expansion of the white pulp with obliteration of normal architecture (Hx. & E. ×100).
On the other hand administration of aflatoxins did not indicate any change in the spleen tissue. Co-administration of honey with aflatoxins causes improvement of all histopathologic changes that occurred in all examined organs except for kidney tissue.
Effect of honey on colonic probiotic bacteria
Addition of honey to AIN-93 M powder diet (HTC group) increased the counts of bifidobacteria by a mean of 2.3 ± 0.23 log10CFU/g (p < 0.004) and lactobacilli by a mean of 1.07 ± 0.35 log10CFU/g (p < 0.016) in comparison to NTC group (Table ).
Effect of honey and mycotoxins on LAB growth (log10CFU/g).
Administration of ochratoxin A in to the group receiving honey (HOT group) increased the counts of lactobacilli by a mean of 0.75 ± 0.15 log10CFU/g (p < 0.108) in comparison to OT group. Lactobacilli were not detected in the case of aflatoxins administration (HAT & AT groups) (Table ).
Administration of ochratoxin A and aflatoxins mixture in presence of honey (HOT & HAT groups) increased the mean counts of bifidobacteria by 1.03 ± 0.32 log10CFU/g (p < 0.083) and 0.57 ± 0.50 log10CFU/g (p < 0.219) in comparison to OT and AT groups, respectively.