THE EFFECT OF PATULIN ON FEMORAL BONE STRUCTURE IN MALE RABBITS

A lot of kinds of crops are susceptible to fungal attack, leading to considerable financial losses and damage the health of humans and animals. Patulin, a toxic fungal metabolite, can be found mainly in apple and apple products, with much less frequent contamination in other food products. Because of its high incidence and harmful health effects, patulin belongs to a class of mycotoxins, which are strictly monitored. However, its effect on bone structure is still unknown. This study was designed to investigate the impact of patulin on femoral bone structure in adult male rabbits. Four month-old male rabbits were randomly divided into two groups of three animals each. Rabbits from the experimental group (group A, n=3) were intramuscularly administered with patulin at dose 10 μg.kg -1 body weight (b.w.) twice a week for 4 weeks. The second group without patulin administration served as a control (group B, n=3). At the end of the experiment, body weight, femoral weight and length, cortical bone thickness and histological structure of femoral bones from all rabbits were determined. The results did not show any significant differences in body weight, femoral weight and length between experimental and control groups of rabbits. On the other hand, intramuscular application of patulin induced a significant increase in cortical bone thickness (p <0.05) and considerable changes in qualitative histological characteristics of compact bone in adult male rabbits. In patulin-intoxicated males, the primary vascular longitudinal bone tissue was absent near endosteal border. On the other hand, this tissue occurred near periosteum and also in the middle part of the femoral bone in these rabbits. The values for the primary osteons’ vascular canals were significantly lower (p <0.05) in males exposed to patulin as compared to the control group. Based on these findings we can conclude that intramuscular patulin administration demonstrably influences cortical bone thickness and histological structure of femoral bone in adult male rabbits.


INTRODUCTION
In the study by Selmanoğlu (2006), some histopathological alternations were observed in the epididymis and prostate tissues in rats orally treated with patulin for 90 days.Also, patulin-induced degeneration and necrosis in liver tissues, as well as degeneration of glomeruli and haemorrhage between the tubules of the cortical region in kidney tissues were reported in male rats (Al-Hazmi, 2012).However, the effect of patulin on bone microstructure had not been studied prior to our experiment.
The aim of the present study was to determine the effect of patulin on selected growth characteristics (body weight, femoral weight, femoral length and cortical bone thickness) and femoral bone microstructure in male rabbits.age of 4 months, weighing 3.5 -4.0 kg) were obtained from an experimental farm of the Animal Production Research Centre in Nitra, Slovak Republic.Male rabbits were used because they are less susceptible to skeletal damage than females (Riggs et al., 2004).Males were housed in individual flat-deck wire cages (area 0.3 m 2 ) under standard conditions (temperature 20 -22 °C, humidity 55 ±10 %, 12/12 h cycle of light and darkness) with access to food (feed mixture) and drinking water ad libitum.
Clinically healthy animals were randomly divided into two groups of three individuals each.In the first group (A), adult rabbits were intramuscularly injected with patulin (10 μg.kg -1 b.w.) two times per week for four weeks.The dose of patulin was estimated based on literature data (FAO, 2004).The second group (B; n=3) without patulin intoxication served as a control.The Animal Experimental Committee of the Slovak Republic approved all procedures.
At the end of the experiments, all the rabbits were euthanized, weighed and their femurs were collected for macroscopical and microscopical analyses.The femurs were weighed on analytical scales with an accuracy of 0.01 g and the femoral length was measured with a caliper.For histological analysis, the right femurs were sectioned at the midshaft of the diaphysis and the segments were fixed in HistoChoice fixative (Amresco, USA).The segments were then dehydrated with a graded series (40 to 100 %) of ethanol and embedded in Biodur epoxy resin (Günter von Hagens, Heidelberg, Germany) according to the method described by The quantitative (histomorphometrical) variables were assessed using the software Motic Images Plus 2.0 ML (Motic China Group Co., Ltd.).We measured area, perimeter and the minimum and maximum diameters of primary osteons' vascular canals, Haversian canals and secondary osteons in all views (i.e., anterior, posterior, medial and lateral) of the thin sections in order to minimize inter-animal differences.Diaphyseal cortical bone thickness was also measured by Motic Images Plus 2.0 ML software.Twenty random areas were selected and average thickness was calculated for each femur.
Statistical analysis was performed using SPSS 8.0 software (SPSS Inc., USA).All data were expressed as mean ± standard deviation (SD).The unpaired Student's T-test was used for establishing statistical significance (p <0.05) between groups A and B.

RESULTS
Our results showed non-significant impact of patulin intramuscular administration on body weight, femoral weight and femoral length in male rabbits.On the other hand, cortical bone thickness was considerably increased (p <0.05) in these animals (Table 1).
Femoral diaphysis of rabbits from the group B had a common bone microstructure.The periosteal and endosteal surfaces were formed mainly by primary vascular longitudinal bone tissue, as a basic structural pattern of all bones.The tissue was created by vascular canals, which ran in a direction essentially parallel to the long axis of the bone.Additionally, primary vascular radial bone tissue (created by branching or non-branching vascular canals radiating from the marrow cavity) was also identified in some areas near the endosteal surface.The middle part of substantia compacta was formed by dense Haversian (characterized by a large number of secondary osteons) or irregular Haversian (characterized by an occurrence of scattered secondary osteons) bone tissues (Figure 1).
In rabbits exposed to patulin, an absence of the primary vascular longitudinal bone tissue near the endosteal surface was found.This part of bone was created only by dense Haversian bone tissue.The periosteal surface was composed of primary vascular longitudinal bone tissue, which also occurred in some areas of the middle part of the compact bone (Figure 2).For the quantitative histological analysis, 249 vascular canals of the primary osteons, 175 Haversian canals and 175 secondary osteons were measured in total.The results are summarized in Tables 2, 3 and 4. We have found that all measured variables (area, perimeter, maximum and minimum diameters) of the Haversian canals and secondary osteons did not differ between rabbits from both investigated groups.On the other hand, the size of the primary osteons' vascular canals significantly decreased (p <0.05) in males from the group A.

DISCUSSION
The results of our study have shown that intramuscular application of patulin had not significant effect on total body weight, femoral weight and length in adult male rabbits.Similarly, no demonstrable alterations in the body weight gain were also reported in rats intoxicated with patulin at a dose of 0.1 mg.kg -1 b.w./day in drinking water for 60 or 90 days (Selmanoglu and Koçkaya, 2004; Selmanoğlu, 2006).Additionally, body weight and weight of various organs (e.g., liver, spleen, thymus, kidney with adrenals and lungs) were unchanged in female mice orally exposed to patulin (at the doses of 0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg.kg -1 b.w.) for 28 days  (1988) studied effects of aflatoxin B1 and T-2 toxin on metabolism of calcium and vitamin D. The young rats were administered daily within 7 days with these mycotoxins at the dose of 0.7 mg.kg -1 and 0.54 mg.kg -1 b.w.Administration of the mycotoxins caused hypocalcemia, decreased the concentration of 25(OH)D3 in blood serum and also lower activity of 25-hydroxylase D3 in liver tissue.In kidney, the activity of 24-hydroxylase tended to decrease.Likewise, the study by Glahn et al. (1991) demonstrated a decreased plasma 25-hydroxy vitamin D and 1,25-hydroxy vitamin D levels in threeweek-old male broiler chickens after five days of aflatoxin treatment.In addition, total plasma calcium tended to be lower.These effects are connected with altered vitamin D and parathyroid hormone metabolism (Devegowda and Ravikiran, 2008).According to Lips (2001), deficiency in vitamin D causes secondary hyperparathyroidism, which leads to increased parathormone production.On the basis of these aspects we suppose that other mycotoxins (including patulin) should also have a similar effect on the concentrations of vitamin D and parathormone, which can be connected with evident changes in cortical bone thickness.
According  (2004).On the basis of these aspects we propose that enlargement of bone tissue on periosteal bone surface could also be associated with patulin-modified increased testosterone concentration.
The results of qualitative histological analysis correspond with those reported by other authors (Enlow and Brown, 1956;Martiniaková et al., 2003;Chrenek et al., 2006).The basic structural pattern of compact bone was primary vascular longitudinal in both groups of rabbits.Also, primary vascular radial and dense Haversian or irregular Haversian bone tissues were identified.In males intoxicated with patulin, an absence of the primary vascular longitudinal bone tissue near endosteal surface can be connected with intensive endosteal resorption due to patulin toxicity.On the other hand, primary vascular longitudinal bone tissue was (in some areas) present not only near the periosteal surface but also in the middle part of the compact bone.This findings point to the enhanced periosteal apposition as a compensative mechanism of bone tissue against patulin-induced bone loss from endosteal surface.Studies with liver cells or liver slices have demonstrated that patulin treatment results in glutathione depletion, which is connected with oxidative stress.Patulin-modified increased intracellular oxidative stress was reported in human embryonic kidney and human promyelocytic leukemia cells (Liu et al., 2007).Bone resorption is the unique function of the osteoclasts (Teitelbaum, 2000).Osteoclasts have shown to be activated by reactive oxygen species (ROS) to enhance bone resorption (Baek et al., 2010).Therefore, we suppose that the absence of primary vascular longitudinal tissue near the endosteal surface can be attributed to oxidative stress-induced intensive bone resorption due to patulin toxicity.
The histomorphometrical measurements showed a significant constriction of the primary osteons' vascular canals in rabbits exposed to patulin.This fact can be related to adverse effect of patulin on blood vessels, which are present in vascular canals of primary osteons found that patulin (at the dose of 1.6 mg.kg -1 b.w.) inhibited acetylcholinesterase and NaK-ATPase in the cerebral hemisphere, cerebellum and medulla oblongata in rats leading to higher levels of acetylcholine in these brain segments.Martiniaková et al. ( 2013) noted that increased levels of acetylcholine in the endothelium of blood vessels subsequently activated muscarinic receptors in the endothelium of vascular and Haversian canals in rats.According to Yoopan et al. ( 2008), the alteration of muscarinic function in blood vessel may contribute to hypertension.The binding of patulin to sulfhydryl groups of acetylcholinesterase might also explain this negative impact of this mycotoxin on the size of the primary osteons' vascular canals in rabbits from the group A.

CONCLUSION
Generally, exceeded recommended level of patulin in fruit juices and other fruit products may cause potential risk in humans, particularly in children.
The current study revealed a significant effect of intramuscular application of patulin at the dose of 10 μg.kg -1 b.w.twice per week for 4 weeks on cortical bone thickness and the size of primary osteons' vascular canals in adult male rabbits.In addition, patulin application induced evident changes in femoral bone microstructure of these animals.
Anyway, our results indicate considerable impact of patulin on macroscopical and microscopical structures of femoral compact bone in adult male rabbits.

Fig. 1
Fig. 1 Microscopical structure of compact bone in rabbits from the group B: 1primary vascular longitudinal bone tissue, 2dense Haversian bone tissue, 3 -primary vascular radial bone tissue.

Fig. 2
Fig. 2 Microscopical structure of compact bone in rabbits from the group A: 1primary vascular longitudinal bone tissue, 2dense Haversian bone tissue.

(
Greenlee and Dunnell, 2010).The results of Broom et al. (1944) revealed a very small, transient vasoconstriction in rabbits after patulin administration at the dose of 0.08 mg.kg -1 b.w.Recent experimental studies have demonstrated the direct action of androgens on the blood vessels.Long-term administration of testosterone may elicit harmful effects, especially vasoconstriction (Akishita and Yu, 2012).Furthermore, patulin has a strong affinity for sulfhydryl groups inhibiting the activity of many enzymes (Selmanoglu and Koçkaya, 2004; Puel et al., 2010; Zbynovská et al., 2013).Magan et al. (2004)

(Llewellyn et al., 1998).
Skeletal growth is the result of complex interplay of nutritional, genetic and hormonal factors.Regarding the hormones, essential roles in normal skeletal development play thyroid and growth hormones.It is known that growth hormone deficiency produces severe generalized failure of osteogenesis (

Table 1
Body weight, femoral weight, femoral length and cortical bone thickness in rabbits injected with patulin (10 μg.kg -1 b.w.; group A) and the control rabbits (group B).

Table 2
Data on primary osteons' vascular canals in rabbits from A and B groups.

Table 3
Data on Haversian canals in rabbits from A and B groups.

Table 4
Data on secondary osteons in rabbits from A and B groups.

Jilka et al., 1999; Burr and Guillot, 2012), which
could partly account for its effect on the cells in the osteogenic layer of the periosteum (

Rhee et al., 2011; Burr and Guillot, 2012).
The benefits of intermittent treatment with parathormone have been shown in experimental animals (