Lactogenic and Cytogenetic Effects of Ochratoxin A in Adult Male Rats and Pups

Lactogenic and cytogenic effects were studied for Ochratoxin (OTA) dosed daily orally throughout lactation period to four groups each consist of newly parturated female rats at doses (0, 60, 120, 180) μg/Kg. BW representing control, T1, T2, T3 group. Micronucleus test results indicated significant increase in number of fragmented and budding nuclei of T1, T2, T3 adult rat bone marrow in dose dependent manner in comparison with control group. The lactating results show no significant change in weekly pup group’s weight gain or length throughout lactating period. Alough there were no changes recorded in viability index of all pups groups, lactating index recorded considerable decline in T1, T2, T3 pups groups according with their adult OTA doses with maximum pups death at the third lactating week. Different histopathological lesions observed in pups liver, kidney and spleen that increase in severity proportionally with their OTA mother doses.


INTRODUCTION
Ochratoxin A (OTA) is a fungal secondary metabolite of mainly Aspergillus ochraceus and Penicillium verrucosum (EFSA, 2004).It has gained great importance due to its biological effects and widespread toxicity.OTA is nephrotoxic and more importantly, it is implicated as a causal factor of Balkan Endemic Nephropathy (BEN) in humans (Grollman and Jelakovi´c, 2007).Much has been written about the possible role of OTA in the etiology of these phenomena and detailed reviews on OTA toxicology have been published (O'Brien and Dietrich, 2005).OTA is thought to be mutagenic, carcinogenic, teratogenic and immunosuppressive in a variety of animal species.It is a mitochondrial poison causing mitochondrial damage, oxidative burst and lipid peroxidation and interferes with oxidative phosphorylation.In addition, OTA increases apoptosis in several cell types (1 1 T Lindsey, 3 T 1 1 T 20023 T ; Bennet and Klich, 2003).OTA hazards are results from its long t½ which in human can be reached to 840 h.Also it was reported to be high accumulative in tissues (liver and kidney) and resistance to heat processing (this toxin tolerate autoclaving for 3 h and 121ºC (Ringo et al., 2010).
OTA has low molecular weight of 403.8, so it can be excreted in bile with enterohepatic circulation ability, also OTA has ability to cross blood brain barrier, placental and lactation transfer in considerable amount (Zhang et al., 2009).There were no enough or lack of data on the lactogenic and cytogentic effects of OTA in nursing pups and adult rats, for such we focus on the following aims in this study.

Aims of study:
• Study cytogenetic effect (Micronucleus test) of Ochratoxin A in bone marrow of dosed adult • Study viability index and lactating indices and toxic effect of OTA in rat pups

MATERIALS AND METHODS
OTA crystalline material (5 mg) was purchased from Sigma that kept by wrapping in aluminum foil at -20°C, avoiding possible degradation.Different concentration of OTA solutions were prepared accordingly for the dose used by dissolving OTA in 2% methanol just before use (Gonzalez et al., 2006).
Experimental animals and design: Twenty mature female albino Wistar rats (150-200 g) were mated at 3-4 month of age.The pregnant females were individually housed and after delivery they were divided equally into four groups each contain five individual animals dosed orally with OTA daily at following doses (0, 60, 120 and 180 µg/kg b.w.) representing control, TR 1 R, TR 2 R and TR 3 R groups with their nearly 205 indirectly dosed suckling pups.

Histopathological preparations and examination:
After scarification of different pups groups at the end of lactation.Specimens of their livers, kidneys and spleens were fixed by 10% neutral buffered formalin solution till the preparation of histological sections.Tissues were embedded in paraffin and several tissue sections were prepared for histopathological examination after staining with Hematoxylin-Eosin (H and E) Stain (Luna and Lee, 1968).

Statistical analysis:
Statistical analysis of the experimental results were conducted according to Statistical Package for the Social Sciences (SPSS) version 13.00 1 T where one way ANOVA was used to assess the significance of changes between control and  (Joda, 2008).

Pups rats body weight change:
The result of body weight change (g/week) in pups rats showed no significant change at (p<0.05) between the treated T1, T2 and T3 groups and between each and control group at zero time, 7, 14 and 21 days of lactation period to dams dosed orally with ochratoxin A for 21 days during lactation.
Between period result indicate significant increase in body weight at (p<0.05) for each control and treated groups positively related with the time, Table 1 and Fig. 1.

Pups rats length:
The result of body length change (cm/week) in pups rats showed no significant change at (p<0.05) between the treated T1, T2 and T3 groups and between each and control group at zero time, 7, 14 and 21 days of lactation period to dams dosed orally with ochratoxin A for 21 days during lactation.
Between period result indicate significant increase in body length at (p<0.05) for each control and treated groups positively collated relationship with the time, Table 2, Fig. 1.
Lactation index recorded as 92.7, 89.8 and 80.8% for T1, T2 and T3 respectively in comparison with 100% for control suckling pups group, Table 4.

Cytogenetic study:
The results of Micronucleus test in adult female dams treated groups showed a significant increase (p<0.05) in number of fragmented and budding  T3 (180 µg/kg OTA): Same as T1 and T2 but more severe lesion was observed with more hyperplasia of white pulp, Fig. 5.

DISCUSSION
Our present study results of OTA recorded no body weight and length changes of rat pups at the end of lactation period after administration of their dams with OTA, this is may be resulted from short time of exposure and the low OTA dam doses but this not exclude its effect on pups survival during lactation accordingly with their dam doses since lactation index were decreased 10 and 20% in T2 and T3 respectively which indicate that the transferred OTA in milk in these groups induce physiological change and pathological lesion in important organs and tissues like brain, liver, spleen and kidney that confirmed by present histological results in organs of indirectly dosed suckling pups.All of these might lead to increase in mortality rate and decline in lactating index of pups groups accordingly with the OTA amount transferred in the milk of their dosed dams which were enough to overcome the low ability to metabolize in liver and excrete by the kidney or by hindering transport through BBB which reported to be not well developed in neonate and pups (Cornford et al., 1982).
The lactating results recorded in the present study are in agreement with Hallen et al. (1998) who investigated the placental and lactation transfer of OTA to pups in cross fostering study in rats that showed OTA at 50 µg/kg did not result in any effects on birth weight or growth development of the pups during the first 21 days of life.In that study, the mean milk: blood ratio of approximately 0.6 was found.The dose of OTA from milk to the suckling pup at 14 days of age calculated to about 50 µg/kg body weight day, which is similar to the dose given to the dams.Pups exposed to OTA only via milk had blood and kidney levels of OTA approximately 3 times higher than their dams, indicating a high absorption and/or a low excretion of OTA in the suckling.At 14 days of age the highest blood and kidney levels of OTA were found in offspring exposed both via placenta and milk, with the highest contribution from milk.This may explain why lactating index showed higher ratio at 2 nd and 3 rd week in our present study.
In other study conducted by Moré and Galtier (1975) on the effects of OTA on dam rats given 2.5 mg OTA/kg bw twice, the mean body weights in male and female offspring at 82 days were reduced by 12 and 8%, respectively.In 26% of male offspring of that group hydrocephalus was observed on day 15 after birth and 40% of these animals died by 20 days after birth.
The micronucleus test result indicates the presence of nuclear genotoxicity in bone marrow cells positively proportional with OTA doses in different females treated groups.This result is in agreement with the finding of Al-Fatlawi (2012) who reported that different doses of OTA caused different chromosomal aberrations and increase in mitotic index in bone marrow cells of adult male rats in dose dependent manner.This may be attributed to the reported genotoxic effects resulted from the oxidative damage on DNA nucleotide bases by OTA in different in vivo and in vitro studies.
In cell culture, an OTA-dependent increase in DNA damage (such as formation of 8-oxoguanine) was correlated with the production of ROS (Schaaf et al., 2002;Kamp et al., 2005).Antioxidants were shown to prevent OTA-mediated increases in MDA production in vitro and in vivo (Baudrimont et al., 1997).In conclusion, the reported transfer of OTA in milk of dosed dams during lactation was at high level at the 2 nd and 3 rd week of lactation period in concentration more than their blood.This may be enough to induce oxidative changes and damage mainly in suckling pups parenchyma tissues like liver, kidney and spleen that are rich in mitochondrial oxidative activities by increasing production of ROS that overcome cellular antioxidant defense mechanism (Baudrimont et al., 1997).

Fig. 1 :
Fig. 1: Rat's pups at the end of lactation period for nursing dams dosed daily orally with OTA at different doses 1 T treated adult rats and their pups.The data were expressed as Mean±Standard Errors (S.E.) and p-value<0.05was considered statistically significance.1 T LSD was carried out to test the significance levels among means of treatments(Joda, 2008).

Fig. 3 :
Fig. 3: Histopathological section of T1, T2 and T3 rat pups liver after 21 days of treatment with OTA doses showed (T1): Vacuolation of hepatocytes ( ) (T2): Few mononuclear cells aggregation around blood vessels ( ) (T3): Aggregation of macrophages ( ) nuclei of bone marrow cells between all treated groups T1, T2 and T3 accordingly with their doses after 21 days from administration of OTA and in comparison with the control group, Table 5, Fig. 2. Histopathological study: Liver: T1 (60 µg/kg OTA): The main lesion characterized by congestion of central veins with vacuolation in the cytoplasm of hepatic cells.T2 (120 µg/kg OTA): Main lesion similar to T1 but more severe lesion observe with few mononuclear cells aggregation around blood vessels.T3 (180 µg/kg OTA): Same but more severe lesion with aggregation of macrophages in the portal area around the blood vessels, Fig. 3. Kidney: T1 (60 µg/kg OTA): Atrophy of glomerular tufts with dilation of bowman's capsule with mononuclear cells

Table 2 :
Body weight change (g/week) of rats pups at end of lactation period for nursing dams dosed daily orally with OTA at different doses

Table 3 :
Viability index of rat pups through lactation period for nursing dams dosed daily orally with OTA at different doses