C 2014 Wiley Periodicals, Inc.

Birth Defects Research (Part B) 98:493–502 (2013)

Review Article

Ochratoxin A: Developmental and Reproductive Toxicity—An Overview Frantisek Malir,1∗ Vladimir Ostry,2 Annie Pfohl-Leszkowicz,3 and Eva Novotna4 1 Department

of Biology, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic for Health, Nutrition and Food, National Institute of Public Health in Prague, Brno, Czech Republic 3 Department Bioprocess & Microbial Systems, INP/ENSA Toulouse, Laboratory Chemical Engineering, UMR 5503 CNRS/INPT/UPS, University of Toulouse, Auzeville-Tolosane, France 4 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University Prague, Hradec Kralove, Czech Republic 2 Center

Ochratoxin A (OTA) is nephrotoxic, hepatotoxic, reprotoxic, embryotoxic, teratogenic, neurotoxic, immunotoxic, and carcinogenic for laboratory and farm animals. Male and female reproductive health has deteriorated in many countries during the last few decades. A number of toxins in environment are suspected to affect reproductive system in male and female. OTA is one of them. OTA has been found to be teratogenic in several animal models including rat, mouse, hamster, quail, and chick, with reduced birth weight and craniofacial abnormalities being the most common signs. The presence of OTA also results in congenital defects in the fetus. Neither the potential of OTA to cause malformations in human nor its teratogenic mode of action is known. Exposure to OTA leads to increased embryo lethality manifested as resorptions or dead fetuses. The mechanism of OTA transfer across human placenta (e.g., which transporters are involved in the transfer mechanism) is not fully understood. Some of the toxic effects of OTA are potentiated by other mycotoxins or other contaminants. Therefore, OTA exposure of pregnant women should be minimized. OTA has been shown to be an endocrine disruptor and a reproductive toxicant, with abilities of altering sperm quality. Other studies have shown that OTA is a testicular toxin in animals. Thus, OTA is a biologically plausible cause of testicular cancer in man. Birth Defects Res (Part B) 98:493–502, 2013.

 C 2014 Wiley Periodicals, Inc.

Key words: ochratoxin A; reprotoxicity; developmental toxicity; teratogenicity; pregnant woman; testicular cancer

INTRODUCTION Ochratoxins are one of the important groups of mycotoxins. In this group, many forms of ochratoxins and their metabolites have been described including phenylalanine-based ochratoxin A (OTA), B, and C (Xiao et al., 1995, 1996; Azpilicueta et al., 2008; Wu et al., 2011; Tozlovanu et al., 2012). Some metabolites such as the lactone-opened OTA (OP-OTA) and OTHQ (quinone form) have also been found in blood and urine, and have been described to be as toxic as OTA (OPOTA) or more toxic OTHQ (Xiao et al., 1996; Li et al., 2000; Faucet-Marquis et al., 2006; Tozlovanu et al., 2006; Pfohl-Leszkowicz, 2009). Among ochratoxins, OTA (see Fig. 1) is the prevalent one. OTA is suggested to contribute to human disease and is a cause of farm animal disease and economic losses (Pfohl-Leszkowicz et al., 2002, 2007; Malir et al., 2012, 2013a) to farmers and food processors. Worldwide, OTA is one of the most common mycotoxins found in foodstuffs (SCOOP, 2002; Woo et al., 2012) of both plant and animal origins. It is produced by fungi of genera Aspergillus and Penicillium (Malir et al., 2013a;

Ostry et al., 2013). The consumption of foodstuffs contaminated by OTA represents a major source of exposure for the general population. In agriculture or in food industry or after water damage, exposure to OTA could be due to dermal contact or inhalation (Degen et al., 2007; PfohlLeszkowicz, 2013). OTA has been shown to have many toxic effects in animals and humans (Malir et al., 2013a). The most important are nephrotoxicity, immunotoxicity, and carcinogenicity. IARC (1993) classified OTA as a possible human carcinogen (group 2B). The susceptibility to cancer is species- and sex-dependent (Castegnaro et al., 1998; Pfohl-Leszkowicz et al., 1998; Pfohl-Leszkowicz and Manderville, 2007; Malir et al., 2013a; Hsuuw et al., 2013). Grant sponsor: Czech Ministry of Health (IGA MZ CR); Grant number: NT 12051-3/2011. ∗ Correspondence to: Frantisek Malir, Department of Biology, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec Kralove, Czech Republic. E-mail: [email protected] Received 2 October 2013; Accepted 10 December 2013 Published online in Wiley Online Library (wileyonlinelibrary.com/journal/ bdrb) DOI: 10.1002/bdrb.21091

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Fig. 1. Chemical structure of OTA.

Even though the mechanism of action remains a controversial topic under debate (Malir et al., 2013a), frequent exposure to OTA can cause health problem. OTA ingestion during pregnancy can affect the intrauterine life (Wangikar et al., 2005). Chronic exposure to low OTA doses could be much more damaging than acute exposure to a high dose (Pfohl-Leszkowicz et al., 2007; PfohlLeszkowicz, 2009). While the reproductive toxicity of OTA is well documented in animals, there are currently no data linking OTA exposure to human reproductive toxicity.

OTA BIOMONITORING No data on teratogenic dose and teratogenic OTA effects in humans are available. The fetal and neonatal exposure to OTA is likely determined by the analysis of cord blood samples from pregnant women, human blood, colostrum, milk, and urine (Micco et al., 1991; Kovacs ˜ et al., 1995; Miraglia et al., 1998; Skaug et al., 1998; Munoz et al., 2010; Klapec et al., 2012). It is clear that OTA fetal exposure might pose a potential risk in prenatal and postnatal life for the human infants (Micco et al., 1991; Jonsyn et al., 1995; Kovacs et al., 1995; Skaug et al., 1998). Since OTA is excreted via breast milk, breast-fed children are also exposed to OTA (Gareis et al., 1988). Although OTA amounts in milk are much lower (10 times less) than those of OTA in blood (Breitholtz-Emanuelsson et al., 1993), OTA contamination of human breast milk presents a possible health hazard (Kuiper-Goodman et al., 2010). The determination of OTA in blood remains the basic method for monitoring human exposure to OTA (Malir et al., 2012). Scott (2005) has described OTA in blood serum as a uniquely useful biomarker of OTA exposure due to its high-affinity binding to serum albumin or other small proteins. Serum OTA contents in pregnant women ranged from 0.06 to 3.41 ␮g/l (Zimmerli and Dick, 1995; Rosner et al., 2000; Postupolski et al., 2006, Malir et al., 2013b). Recently, a study on the assessment of OTA intake by pregnant women in the first trimester of pregnancy was conducted. Serum OTA amounts ranged from 0.1 to 0.35 ␮g/l with a mean value of 0.15 ␮g/l. Presence of OTA in serum of pregnant women correlates with OTA dietary exposure assessment (Malir et al., 2013b). In utero, exposure of human fetuses to OTA is proven by the presence of OTA in fetal serum and cord blood samples from 0.13 to 5.42 ␮g/l (Zimmerli and Dick, 1995; Postupolski et al., 2006; Biasucci et al., 2011). This finding

raises concerns about potential health hazards to human fetuses. OTA passing through placenta via active transport (Zimmerli and Dick, 1995; Postupolski et al., 2006) is found in human fetal samples (Jonsyn et al., 1995; Zimmerli and Dick, 1995; Postupolski et al., 2006; Biasucci et al., 2011). The higher amount of OTA in fetal serum than in maternal serum (Zimmerli and Dick, 1995; Postupolski et al., 2006) is due to gradual accumulation over chronic exposure during early gestation (Zimmerli and Dick, 1995). OTA determination in urine is studied to clarify the relationship at the individual level between OTA intake and urinary biomarker (Duarte et al., 2011). OTA in urine is a promising alternative to the measurement of OTA in blood, even though the OTA concentration in urine is much lower than in blood. Despite these difficulties, OTA in urine is thought by some authors to be a better indicator of exposure to OTA than OTA in plasma (Gilbert et al., 2001; Castegnaro et al., 2006; Pfohl-Leszkowicz et al., 2006). Moreover, analysis of OTA metabolites in urine will give additional information concerning toxicokinetic and genetic susceptibility (Pfohl-Leszkowicz, 2009). The first study to determine the exposure of pregnant women to OTA was realized through examination of OTA and ochratoxin alpha in the urine of pregnant women (Klapec et al., 2012).

OTA AND PLACENTA Human placenta plays an important role in the development of the fetus, being the major interface between mother and fetus. Placenta provides a gateway to oxygen and nutrients from the mother to the fetus. It produces hormones to support the pregnancy and serves as an excretion pathway for various metabolites and carbon dioxide. However, it also plays a role in the exposure of the fetus to potential xenobiotics. Transport processes across the placenta (passive diffusion, active transport, facilitated diffusion, filtration, and pinocytosis) and metabolism determine the exposure of the fetus to xenobiotics including OTA (Myllynen and V¨ah¨akangas, 2013). Very little is known about the mechanism of OTA transfer across human placenta. Presence of transporters, immature metabolism, and low elimination capacity of the fetus contribute to the bioaccumulation of OTA in the fetal circulation (Woo et al., 2012). The transfer of OTA is limited by its high-affinity binding to plasma proteins (Woo et al., 2012). It has been estimated that approximately 99% of the circulating OTA is bound to plasma proteins (mainly albumin; Chu, 1971, 1974; Dai et al., 2004) and only a small amount of OTA occurs freely. In human perfused placenta, OTA transfer is not concentrationdependent (Woo et al., 2012). This can be explained by the fact that only unbound molecule is capable of passing through placental barrier (Loebstein and Koren, 2002). OTA administered to pregnant rats is detected in embryos (Mor´e and Galtier, 1978a; Appelgreen and Arora, 1983a; Ballinger et al., 1986; Fukui et al., 1987; Hallen et al., 1998; Minervi et al., 2013). At the beginning of the pregnancy, OTA quickly passes through mouse placenta (Appelgreen and Arora, 1983b). After crossing the maternal placental barrier, OTA reaches the fetus and Birth Defects Research (Part B) 98:493–502, 2013

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interferes with the development of the organs (Appelgreen and Arora, 1983b). A study on OTA in human perfused placenta shows that OTA is able to cross the placenta, more particularly, at early gestation rather than at late gestation (Woo et al., 2012) Direct genotoxic effect of OTA, reflected by DNA adducts, in the progeny (the fetus and the offspring) through transplacental contamination was demonstrated in a mice study. High amounts of DNA adducts were detected in the kidneys of male progeny, and in liver of female progeny and their mothers exposed to a single-OTA dose during late pregnancy (Petkova-Bocharova et al., 1998b). These data correlate well with the carcinogenicity in mice, for which the target organ for males is the kidney, while for females it is the liver (Petkova-Bocharova et al., 1998b). Similar data were obtained with hamster (Petkova-Bocharova et al., 1998a). Some owners and breeder of cats and dogs observed a decrease of fertility and stillbirth when the animals are fed with petfood. Cereal grain and nuts are often used as ingredients in industrial petfood. All the pet food, leading to reproductive problems, was contaminated by several mycotoxins. The petfood without cereal contains much less mycotoxins and enabling normal birth. Large amount of OTA, zearalenone, fumonisins B, aflatoxin, and their metabolites were found in blood, kidney, liver, intestine, brain, and placenta of monstrous cat. Specific DNA adducts (related to OTA and/or zearalenone (ZEA)) were detected in the tissue of dead little cat. The amounts of individual mycotoxins are low, but their simultaneous occurrence was responsible for the reduced fertility and stillbirth because these mycotoxins have a synergistic effect (Hadjeba-Medjdoub et al., 2013). The continuous exposure to OTA—especially during the organogenesis period—might be responsible for the microscopic changes observed in vital organs of developing fetuses and also for the histopatologic alterations in the fetal organs of rabbits (Wangikar et al., 2005).

genase activity and reduces ovarian steroidogenesis in rats (Gupta et al., 1980). In human placenta, 3␤-HSD1 is expressed at high levels in synctial trophoblast. It catalyzes the conversion of 3␤-hydroxy-5-ene-steroids (dihydroepiandrosterone, pregnolone) to main 3-oxo-4-enesteroids (androstendione, progesterone) to maintain the uterus in a quiescent state (Thomas et al., 1989; Kacsoh, 2000). Androstendione is then converted by aromatase and 17␤-hydroxysteroid dehydrogenase (17␤-HSD) to estradiol. As 3␤-HSD1 is exclusively expressed in human placenta and plays an important role in placental steroidgenesis, modulation of placental 3␤-HSD1 activity may result in disturbance of steroid production and hence affect placental and fetal development (Woo et al., 2013). To study the effects of short- and long-term OTA exposure on the expression of 3␤-HSD1 and progesterone secretions, human placental cells human choriocarcinoma cell line (JEG-3) were exposed to OTA. It was shown that OTA upregulates 3␤-hydroxysteroid dehydrogenase type 1 expression in human placental cells. The induction of 3␤HSD1 mRNA leads to an increase in progesterone production (Woo et al., 2013). OTA also induces the production of estradiol (Frizzell et al., 2013). These data can partially explain the impact of OTA on kidney. Indeed, it has been demonstrated that endogenous sex steroid 17␤-E2 reduces renal basolateral organic cation transport through direct action on renal tubule cells (Pelis et al., 2007). In contrast, OTA inhibits testosterone secretion in vitro (Fenske and Fink-Gremmels, 1990). In rat, OTA inhibits both cortisol and testosterone secretions (Kumar et al., 2011). In another rat study, OTA induces folliclestimulating hormone, testosterone, triiodothyronine 3, and thyroxin, and reduces luteinizing hormone (Hassan et al., 2010). OTA and endosulfan (a pesticide) synergystically alter the hormonal status of male Wistar rats (Kumar et al., 2011).

OTA AND REPROTOXICITY

Male reproductive health is an important component of men’s overall health and wellbeing. Too often, males have been overlooked in discussions on reproductive health, especially when reproductive issues such as contraception and infertility have been perceived as femalerelated. Every day, men, their partners, and healthcare providers can protect their reproductive health by ensuring effective contraception, avoiding sexually transmitted diseases, and preserving fertility. Erectile dysfunction, premature ejaculation, loss of libido, testicular cancer, and prostate disease may cause embarrassment to the patient and, occasionally, the general practitioner (Wijesinha et al., 2013). Male reproductive health has deteriorated in many countries during the last few decades. Several toxins in environment have been suspected to affect reproductive system in male, and OTA is one of them (Verma and Chakraborty, 2008). Male fertility can be affected by impairment of sperm production and/or damage of testis. In mice, OTA exposure significantly decreases sperm count and motility and increases abnormalities in sperm morphology (Bose and Sinha, 1994; Biro´ et al., 2003; Farag et al., 2010). OTA (50 and 100 ␮g/day) given per os to male albino mice alters sperm count, sperm motility,

OTA and Endocrine Disruption All biologic functions in organism are controlled by hormones. Consequences of endocrine disruption are reproductive disorders (decreased libido, disturbed reproductive cycle, anovulation, disturbed spermatogenesis, infertility, neoplasmic lesion, altered behavior, decreased fertility of offspring), osteoporosis, myelofibrosis, adenoma, and skeletal deformations (Anonymous, 2013). Some data show that OTA can act as an endocrine disruptor by interfering with enzymes involved in the synthesis of the steroids or hormones. OTA does not interact directly with the receptor as it is the case for zearalenone, another mycotoxin (Kuiper-Goodman et al., 1987; Zinedine et al., 2007). The enzymatic complex 3␤-hydroxysteroid dehydrogenase (3␤-HSD) is located in the endoplasmic reticulum and mitochondria. Complex 3␤-HSD plays an essential role in the biosynthesis of all classes of steroid hormones. It catalyzes the oxidation and isomerization of 5– 3␤hydroxysteroid precursors into 4 -ketosteroids, first step in the biosynthesis of androgens and estrogens (Thomas et al., 1989). OTA suppresses 3-hydroxysteroid dehydroBirth Defects Research (Part B) 98:493–502, 2013

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sperm viability, and fertility rate in a dose-dependent way (Chakraborty and Verma, 2009). OTA also reduces the fertility of pigs. Indeed, OTA was found in seminal liquid of boar exposed by feed to 20 to 40 ␮g of OTA (Solti et al., 1999; Biro´ et al., 2003). The sperm production and semen quality of boars were decreased by OTA, because OTA provokes instability of the spermatozoal membrane (Ewald and Heer, 1989; Solti et al., 1999). These effects are more pronounced when pigs are simultaneously exposed to OTA and zearalenone (Posea et al., 2013).

OTA and Testicular Cancer In animals, OTA is toxic for testis (Mor´e and Camguilhem, 1979, Gharbi et al., 1993; Bose and Sinha, 1994; Biro´ et al., 2003). Several areas of the Balkans that are known to have high incidence of OTA contamination of food have recently experienced a marked increase in the incidence of testicular cancer. For example, from 1960 to 1985, the incidence of testicular cancer in Vas, Hungary, increased fivefold, from 1.2 to 6.1/100,000 (Huyghe et al., 2007). A similar increase was reported for Slovenia (Huyghe et al., 2007). A possible etiologic role for OTA in testicular cancer in men was hypothesized in 2002 by Schwartz, who noted that the unusual geographic distribution of testis cancer, with especially high rates in Denmark, was correlated with the national per capita consumption of OTAcontaminated foods. Recent toxicologic data in rodents support the concept that dietary exposure to OTA can influence carcinogenesis in the testis. For example, Ueta et al. (2010) administered 2 mg/kg OTA intraperitoneally to Pdn/Pdn mice on day 7.5 of gestation. They observed a significant downregulation of Dmrt-I gene expression in the male conceptuses on day 9 (Ueta et al., 2010). Dmrt-1 (also known as doublesex and mab-3–related transcription factor 1) is essential for the normal development of the mammalian testis (Raymond et al., 2000). DMRT1 is expressed in Sertoli cells and undifferentiated spermatogonia of the postnatal testis (Murphy et al., 2010). DMRT1 is a tumor suppressor gene in the testis; loss of this gene produces germ cell testicular tumors in mice (Krentz et al., 2009) and humans (Turnbull et al., 2010). More direct evidence of a carcinogenic role of OTA in the testis was provided by Mantle and Nolan (2010). These investigators administered 100-␮g OTA as a daily dietary contaminant to 24 male Fischer rats for 35 weeks. Testis tumors developed in six of the rats (25%), an incidence rate equal to that of renal tumors in male Fischer rats chronically exposed to dietary OTA. In a recent study, DNA damage to testis was proved experimentally by intrauterine exposure to OTA (2.5 mg/kg/bw) at day 17 of the gestation. DNA adducts in the testes of newborn mice are similar to the DNA adducts observed in the kidney and testes of adult mice exposed to OTA via the diet (Jennings-Gee et al., 2010). DNA adducts in the testes of mice exposed prenatally to OTA, and the absence of any such adducts in the testes of control mice not exposed prenatally to OTA, are clear evidence of the carcinogenic potential of OTA in the testes. The DNA adducts that we observed are not evidence merely of exposure (as Mantle (2010) suggests), they are markers of biologic effect, as DNA adducts are widely

considered to be markers of an increased risk of cancer (Bonassi et al., 2001; Knudsen and Hansen, 2007; PfohlLeszkowicz, 2008). Schwartz et al. (2010) substantiated the finding reported in Jennings-Gee et al. (2010) by explaining the recent research that has shown that prenatal exposure to OTA in mice significantly depresses the expression of the DMRT1 gene in offspring, particularly in male offspring (Ueta et al., 2010). Hence, significant molecular evidence supports the hypothesis that OTA may be causally related to germ cell testicular tumors in mice and in men. Future epidemiologic and molecular studies in humans are needed to draw a clear conclusion whether OTA is a causative agent in testicular cancer (Srinavasa, 2011).

OTA and Teratogenicity Teratogenicity is the ability to cause developmental anomalies in a fetus. Teratogens include viruses, chemicals, and radiation (Speight and Holford, 1997). The factors that determine the effects of teratogens include dose reaching fetus, step of the development when toxin exposure occurs, duration of exposure, environmental factors, and susceptibility of the fetus (Young and Koda-Kimble, 1995; Di Piro et al., 1997; Speight and Holford, 1997). The most common developmental toxicities are spontaneous abortion, congenital malformations, intrauterine growth retardation, mental retardation, carcinogenesis, and mutagenesis (Behrman, 1996; Di Piro et al., 1997; Speight and Holford, 1997; Hansen and Abbott, 2009). It is known that the gestation period is divided into several periods and that the fetus is more vulnerable during the period of organogenesis (i.e., in humans between 3 and 8 weeks after fertilization; in rats between 6 and 15 days and so on) with the increasing risk of spontaneous abortion and congenital malformations (congenital defects) of the fetus (Porter, 2004; Patil et al., 2006).The teratogenic effects of OTA have been well documented in many animal models (see Table 1). The reduced birth weight and craniofacial abnormalities are the most frequent signs observed (Patil et al., 2006). Most of the available data on OTA teratogenicity (developmental toxicity) come from rodent studies. Several studies have been conducted after intraperitoneal or subcutaneous administration (Hayes et al., 1974; Gilani et al., 1978; Mayura et al., 1982, 1984b; Wei and Sulik, 1993). A single subcutaneous treatment (1.75 mg OTA/kg) given on day 6 of gestation induces visceral and skeletal malformations in rat fetuses (Mayura et al., 1982). Dietary protein deficiency increases the susceptibility of the animal to the teratogenic effects of OTA notably skeletal development (Mayura et al., 1983). A single dose (2.5–20 mg OTA/kg bw) administrated by intraperitoneally (ip) to pregnant golden hamsters increases prenatal mortality between seventh and tenth day of gestation and diminishes fetal growth on the ninth day. Malformations such as micrognathia, hydrocephaly, short tail, oligodactyly, syndactyly, cleft lip, micromelia, and heart defects occurrs (Hood et al., 1976). Harmful effects of OTA are potentiated by other mycotoxins or contaminants. In particular, in mice treated intraperitoneally, OTA enhances the incidence of fetal malformations induced by T-2 toxin (Hood et al., 1978). Similarly subcutaneous Birth Defects Research (Part B) 98:493–502, 2013

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Table 1 Animal Teratology Studies Animal model Rat

Mice Hamster Chicken embryo Pig Quail Rabbit

Reference Still et al. (1971), Mor´e and Galtier (1974), Mor´e and Galtier (1975), Brown et al. (1976), Mor´e and Galtier (1978a, 1978b), Mayura et al. (1982, 1983, 1984a), Wei and Sulik (1993), Abdel-Wahhab et al. (1999), Wangikar et al. (2004a, 2004b) Hayes et al. (1974), Arora (1982), Petkova-Bocharova et al. (1998a), Katagiri et al. (2007), Jennings-Gee et al. (2010), Ueta et al. (2010) Hood et al. (1975, 1976), Petkova-Bocharova et al. (1998b) Gilani et al. (1978), Wiger and Stormer (1990), Wei and Sulik (1996), Lalitha Kunjamma and Nair (1997) Shreeve et al. (1977) Dwivedi (1984) Wangikar et al. (2004c, 2005)

administration of OTA and citrinin to rats increases prenatal toxicity and teratogenicity (Mayura et al., 1982). Nevertheless, first attention should be given primarily to oral intake, which appears to be natural and realistic way for reliable assessment of developmental disorders induced by food-borne mycotoxins such as OTA (Arora, 1982). Oral administration of OTA (5 mg/kg bw during 2 and 4 days) to the pregnant rats causes a reduced weight of fetus as well as a significant proportion of fetus with hemorrhages. In the F1 generation, there was a reduction in litter size and growth retardation, but no effects were proven in the F2 generation (Mor´e and Galtier, 1974, 1975, 1978b). In another study, oral administration of OTA (0.75 mg/kg bw) to pregnant rats between sixth and fifteenth day of pregnancy induces teratogenicity including visceral anomalies and skeletal anomalies, in addition to embryotoxicity (Brown et al., 1976). Dosing of pregnant rats per os (po) with 0.75 to 1 mg/kg OTA resulted in high incidence of embryo resorption, but also decreased fetal size and increased skeletal defects, primarily in the craniofacial region (Mayura et al., 1982, 1984a, 1984b). External hydrocephaly, omphalocele, and anophthalmia were the major gross anomalies. Internal hydrocephaly and shift in position of esophagus were the main internal soft tissue defects. Major skeletal defects involved sternebrae, vertebrae, and ribs (Mayura et al., 1982). Malformations included growth retardation, hypoplasia of the telencephalon, poor flexion, stunted limb bud development, underdeveloped sensory primordia, and decreased mandibular and maxillary size. Histologic examination demonstrated extensive necrosis of embryonal mesodermal structures and neuroectoderm (Mayura et al., 1989). OTA at different concentrations (2–4 mg/kg bw) and different gestations days (6–15) causes variable developmental defects (external hydrocephaly, incomplete closure of skull, and omphalocele) in fetuses (Patil et al., 2006; van Dorp et al., 2010). A single oral dose of 2.75 mg/kg bw was found as the minimum effective teratogenic dose in pregnant Wistar rats (Patil et al., 2006). The most critical gestational period for the induction of teratogenicity in rats was between the sixth and seventh day (Patil et al., 2006). Craniofacial malformations (exencephaly, midfacial clefting, and cleft lip) were found in

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mice exposed to OTA (Wei and Sulik, 1993). Microcephalic brains in mice exposed to OTA are the consequences of a reduced dendritic growth (Fukui et al., 1992). Altogether, OTA is teratogenic in many animals including rat (Brown et al., 1976), mice (Arora et al., 1983), hamster (Hood et al., 1976), rabbit (Wangikar et al., 2004c), and chick (Wiger and Stormer, 1990). In summary, OTA provokes exencephaly, incomplete closure of skull, hypognathia, micromelia, scoliosis, curled tail, skeletal defects involving fused, bifurcated ribs as well soft-tissue anomalies like the ectopic or polycystic kidney, renal agenesis, hydrocephaly, and microphthalmia (Brown et al., 1976; Mayura et al., 1982; Wangikar et al., 2004b, 2004c). The severity and extent of various gross, skeletal, and visceral anomalies were proportional to the doses applied (Wangikar et al., 2004b). Both OTA and OTB (the dechloro analog of OTA) cause craniofacial malformations, while OTA also causes reduced embryo growth. As expected, OTA is by far more potent in inducing these effects than OTB (O´Brien et al., 2005). OTA induces neural-tube defects (NTDs) in rodent embryos (Wei and Sulik, 1993; Wangikar et al., 2004a) due to the inhibition of protein synthesis (Monnet-Tschudi et al., 1997; Wangikar et al., 2007). An antagonism between OTA and aflatoxin B1 (Wangikar et al., 2004a, 2004b) or between OTA and zearalenone is observed in mice exposed by oral route (Arora, 1982). OTA and aflatoxin B1 (AFB1 ) both interfered with the neural-tube development. However, when aflatoxin B1 and OTA are combined together, the effects on the neural tube decreased, but heart defects arose (Wangikar et al., 2007). In ovo inoculation of OTA in chicken embryos and subsequently in the hatching chicks induces teratogenic defects depending on the dose. The symptoms observed were anophthalmia (49% of the embryos), mandibular hypoplasia (45%), micropthalmia (35%), maxillary retrognathism (12%), reduced body size (15%), everted viscera (8%), spina bifida (10%), and exencephaly (4%; Hassan et al., 2012).

OTA and Molecular Mechanisms of Teratogenicity To define the mechanism of action an in vitro teratogen assay using rat embryo midbrain micromass cultures was

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conducted (Walum and Flint, 1990). OTA induces cytotoxicity of all brain cells without any selectivity against neurons or astrocytes (Wilk-Zasadna and Minta, 2009). Morphometric assessment using computer imaging revealed that OTA teratogenic potency was correlated with their effects on aggregation of neuronal cells, and formation of neurospheres (Wilk-Zasadna and Minta, 2009). The gene responsible for the polydactyly/arhin encephaly (Pdn/Pdn) mouse, which exhibits polysyndactyly and arhinencephaly and has a 13.2% risk of NTDs, has been identified as Gli3. When Pdn/Pdn embryos were exposed to 2 mg OTA/kg administered intraperitoneally to the Pdn/+ females on day 7.5 and/or 7 of gestation, the incidence of NTD in Pdn/Pdn increased from 51.6% (Katagiri et al., 2007). Downregulation of the Gli3 gene induces the overexpression of the Fgf8 gene at the anterior neural ridge and of Emx2 gene in the dorsal forebrain in the Pdn/Pdn mice (Katagiri et al., 2007). Fgf8 is a growth factor important for the development of the telencephalon (Garel et al., 2003; Storm et al., 2003). Emx2 is homeobox gene (Simeone et al., 1992) playing and important role in the morphogenesis of the telencephalon, diencefalon, and mesencephalon (Suda et al., 1996; Acampora et al., 1997; Yoshida et al., 1997; Suda et al., 2001). OTA treatment accelerated the overexpression of these genes. Pretreatment with folic acid ameliorated the OTA-induced overexpression of Fgf8 in the Pdn/Pdn (Katagiri et al., 2007). Exposure to OTA led to increased embryolethality manifested as resorptions or dead fetuses. OTA had the same effect on all genotypes (Pdn/+; Pdn/Pdn; Katagiri et al., 2007). It was concluded that the increase of NTDs incidence could be consequence of OTA toxicity. More recently, gender-dependent differences in the incidence of NTDs induced by OTA were investigated in Pdn/Pdn mouse (Ueta et al., 2010). In this study, on the basis of real-time polymerase chain reaction analyses, it has been suggested that the manifestation of NTDs in the male OTA-treated Pdn/Pdn might be due to the complicated altered gene expressions among Gli3, Wntb7b, Wntb8b, Fez1, Barx1, Lim1, Dmrt1, Igf1, Fog2, Dax1, and Sox9, and in particular, upregulation and genderdependent difference in Barx 1 and gender-dependent difference in Sox9 gene expressions are noteworthy findings (Ueta et al., 2010). Moreover, prenatal exposure of mice to OTA also significantly downregulates the expression of the DMRT 1 (tumor suppressor gene) in male offspring (Ueta et al., 2010). In mice, it has been also shown that a single-OTA dose (0.5 or 2 mg/kg bw) given by gastric intubation to the mother on the 17th or 18th day of gestation induced OTA-DNA adducts in the kidney and liver of fetus and in newborn several months after birth, and in some of them later cancer tumors occurred (Petkova-Bocharova et al., 1998b). Similar results were obtained with hamster (Petkova-Bocharova et al., 1998a). Recent studies showed that in vitro exposure to OTA triggers apoptosis and retards early postimplantation development after transfer of embryos to host mice (Suda et al., 2001). In addition, OTA induces apoptosismediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-

dependent apoptotic signaling processes that impair subsequent embryonic development (Bouaziz et al., 2011). The risk of spontaneous abortions associated with the exposure to endogenous substances may be modified by the genetic variation in individual metabolic detoxification activities, thus, in the phase I/phase II balance (Wang et al., 1998; Zusterzeel et al., 2000). The occurrence of the glutathione-S-transferase P1b-1b genotype, leading to lower glutathione-S-transferase Pi enzyme activity and, consequently, impairing placental detoxification, may represent a risk factor for recurrent early pregnancy loss (REPL; Zusterzeel et al., 2000). In a recent toxicogenomics study, the predominant effect of OTA was downregulation of RNA and protein expressions (Marin-Kuan et al., 2006). OTA is metabolized by conjugation with glutathione (Pfohl-Leszkowicz et al., 1993; El Adlouni et al., 2000; Pfohl-Leszkowicz et al., 2002; Tozlovanu et al., 2012). Presence of the GSTP 1b-1b genotype, associated with impaired detoxification, causes an imbalance between phase I and phase II of biotransformation, and therefore provokes the development of REPL. Thus, homosigoty for the GSTP 1b allele might be associated with an increased risk for REPL (Zusterzeel et al., 2000). Several data also pointed the implication of glutathione in OTA genotoxicity (Pfohl-Leszkowicz and Manderville, 2007; PfohlLeszkowicz, 2008; Pfohl-Leszkowicz and Manderville, 2012). Andonova et al. (2004) have shown that carriers of at least one glutathioneS-transferase M1 (GSTM1) wildtype allele (positive conjugators) were more prevalent among Balkan endemic nephropathy (BEN) patients compared to controls (OR = 7.92).

CONCLUSIONS AND RECOMMENDATIONS More than 1200 chemical and physical agents are known to cause structural and/or functional malformations in experimental animals (Shepard, 2001). Although the mechanisms by which these agents disrupt the normal development are not well understood, it is known that many teratogens induce cell death in tissues that subsequently develop abnormally causing structural malformations (Knudsen, 1997). These observations apply to OTA teratogenicity as well. In addition to be carcinogenic, OTA has been shown to be an endocrine disruptor and a reproductive toxicant, with abilities of altering sperm quality (Biro´ et al., 2003; Anonymous, 2013; Woo et al., 2013). Taking into account the ubiquitous OTA occurrence in food and feed, its long half-life, and important toxicity of OTA and its metabolites (e.g., the OP-OTA, as toxic as OTA; OTHQ, more toxic than OTA; genotoxicity, carcinogenicity and in animals clearly demonstrated teratogenicity; Xiao et al., 1996; Li et al., 2000; Faucet-Marquis et al., 2006; Tozlovanu et al., 2006; Pfohl-Leszkowicz, 2009), risk management should be implemented. Health Canada claims that OTA should be regulated as a nonthreshold carcinogen and the virtual safety dose (corresponding to negligible cancer risk) should be set above 4 ng/kg bw/day (Kuiper-Goodman et al., 2010). In addition, the harmful effects of OTA can be potentiated by possible synergistic action with other mycotoxins or contaminants. In light of the above-mentioned OTA toxic

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REPROTOXICITY OF OCHRATOXIN properties and effects for the unborn child, OTA exposure of pregnant women should be kept as low as possible with an aim of the best protection.

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