Table of Contents  

Abdulrazzaq, Padmanabhan, Shafiullah, Kochiyil, and Bastaki: Intrauterine growth restriction and impaired skeletal growth in mouse fetuses following maternal exposure to aflatoxin B1 during early and late organogenesis

Introduction

Significant exposure of humans to aflatoxins occurs via contaminated food13 (particularly in tropical and subtropical areas) and by air, particularly in farm workers.47 Aflatoxins are a group of closely related mycotoxins that are hepatotoxic, mutagenic and carcinogenic.8 Immunosuppressive metabolites are produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, and four are toxins, namely aflatoxin B1 (AFB1), aflatoxin B2, aflatoxin G1 and aflatoxin G2. AFB1 is of particular concern because it is known to induce hepatocellular carcinoma. In its natural state AFB1 is non-toxigenic, but it becomes harmful when it is converted into its reactive epoxide by the action of cytochrome P450-dependent, mixed-function, mono-oxygenase enzyme systems in the liver.9 In the presence of AFB1, lipids infiltrate hepatocytes, causing hepatocyte necrosis. Consequently, the patient suffers liver dysfunction, derangement of blood-clotting mechanisms, icterus and a reduction in essential serum proteins. Even with low levels of aflatoxin in the diet, immunosuppression is caused10 as aflatoxins inhibit nucleic acid and protein synthesis in addition to decreasing lipid metabolism and mitochondrial respiration.

An extensive literature provides evidence on the acute and chronic toxicity, carcinogenicity and immunosuppressive effects of AFB1, but the results of experimental studies on the effects on reproduction of mycotoxins in general, and aflatoxins in particular, are heterogeneous. Among the reports that are available, there is evidence to show that aflatoxins cross the placental barrier rapidly,1114 with the rate of transfer appearing to be dependent simply on the concentration gradient.15,16 Consequently, it is not surprising to find that administration of AFB1 to mice mid-gestation has also been reported to result in a moderate reduction in maternal body weight and a dose-dependent increase in the number of skeletal and visceral malformations in the fetus, including cleft palate, wavy ribs and diaphragm changes.17,18 Similarly, continuous dosing with a low dose of AFB1 equivalent to 1p.p.m. given intraperitoneally (i.p.) in the first 14 days of gestation was found to result in maternal liver pathology, a reduction in fertility, fetal malformations and developmental delay in rats.15

Aflatoxins are potent hepatocarcinogens in animal models and suspected carcinogens in humans. AFB1 causes cancer by inducing DNA adducts, which lead to genetic changes in the target cells, as a result of which it then causes breakage of the DNA strand, damage to the DNA base and oxidative damage that may ultimately lead to cancer. DNA adducts are formed from chemical modification of the bases in DNA or amino acids in proteins by toxic carcinogenic chemicals.19 Approximately half of human cancers are a result of a mutated TP53 gene. Mutations, such as the transversion in codon 249 (guanine to thymine), which causes an arginine to serine substitution, are present in 50% of hepatocellular carcinomas.20,21

The interspecies differences in AFB1 metabolism are very important in determining its toxic effects. In the human liver, CYP3A4 plays an important role in biotransforming AFB1 to the toxic product AFB1-8,9-epoxide. AFB1-8,9-epoxide could conjugate with glutathione to reduce its toxicity via glutathione-S-transferase (GST). In poultry species, CYP2A6, CYP3A37, CYP1A5 and CYP1A1 are responsible for bioactivation of AFB1. The rate of AFB1-8,9-epoxide formation and its conjugation with glutathione is a key parameter in interspecies differences in sensitivity to the toxic effects of AFB1.22 Mice have a tremendous capacity to conjugate AFB1 via GSTs, whereas rats are more sensitive to AFB1, ultimately culminating in hepatocellular carcinomas. The results of one study23 showed that AFB1 induced liver injury, indicated by an elevated relative liver weight and increased activities of alanine aminotransferase and/or aspartate aminotransferase, as well as decreased albumin and/or total protein concentration in the serum. This mycotoxin also decreased hepatic total antioxidant capacity and/or increased the concentration of malondialdehyde. Furthermore, there was upregulation of the apoptotic genes CASP3 and BAX, along with downregulation of the antiapoptotic gene BCL2 in the liver.

The results from some studies have also indicated that AFB1 is teratogenic to both hamster and chick fetuses16,24 in addition to mice and rats, as reported above. However, a review of the available data indicates that some come from studies that give rise to considerable methodological concerns, particularly in relation to preparation and administration of the water-insoluble test substance, rendering the derived data inconclusive. Different models have been used and it is difficult to compare results between them, especially between avian and mammalian models and between in vitro to in vivo studies. Different dosing regimens have also been used, and the administration of multiple doses of the various toxins and consequent enzyme induction further complicates interpretation of the available data.25 It is clear that the effects of AFB1 on fetal growth have not been adequately investigated. Consequently, this project used the rodent model as in rats prenatal growth is most marked during the last third of gestation.26 However, it is important to note that prenatal growth is a continuum and that organogenesis is accompanied by substantial growth in fetus body size. Accordingly, this study was undertaken to determine whether aflatoxins administered at early and late stages of organogenesis have deleterious effects on general fetal development and growth.

Materials and methods

Animal breeding

Theiler outbred mice were obtained from Envigo (Alconbury Huntingdon, UK) and a colony was raised in a local facility. All mice were housed in a 12 : 12 hours light–dark cycle in temperature-controlled rooms (22 ± 1°C). All mice had access to both a commercial laboratory chow diet and tap water ad libitum. Females weighing approximately 30 g and aged approximately 6 weeks were mated overnight with similar males. The presence of a vaginal plug observed the following morning was regarded as an indication of mating. The plug-positive day was designated gestation day (GD) 0.

Aflatoxin B1 administration

Aflatoxin B1 (from A. flavus, molecular weight 312.27) and reagents were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). For animal treatment, a lyophilized powder of AFB1 was dissolved in dimethyl sulfoxide (DMSO) at 5.0 mg/ml, stirred well and used immediately.

A single i.p. dose of 20 mg/kg body weight AFB1 was administered to groups of mice on either GD 7 or GD 13. A similar dose of AFB1 was given orally through an orogastric tube to different groups of mice also on GD 7 or GD 13. This dosage was selected as we had previously worked with this dose,26 and doses less than this did not have any effect on the fetus. The corresponding control groups consisting of identical mice were administered a similar volume of DMSO, either i.p. or orally.

Maternal body weight and food and water consumption were recorded daily until the animals were sacrificed by cervical dislocation on GD 18. Immediately, the uterine horns were incised along the antimesometrial margin and all available fetuses delivered. The number of implantations, resorptions and fetal deaths were recorded following simple visual observation. Six control and experimental animals were used for each time point in each group in the study. Placental weight and body weight were recorded for each fetus separately and grouped according to litter. The fetuses were fixed and stored separately in 95% ethanol and subsequently observed with a stereomicroscope to evaluate both internal and external malformations.27 Visceral evaluations were made by autopsy. To facilitate this evaluation, the methods of Wilson25 and of Sterz and Lehmann28 were used after slight modification. Morphological evaluations were undertaken on all fetuses; however, skeletons were studied by random selection. The fetuses were subsequently eviscerated and their skeletons stained for cartilage and bone in accordance with the methods described by McLeod.29 The fetuses were processed and stained using alizarin red-S and Alcian blue30 to detect bone and cartilage malformations. These specimens were examined using a stereomicroscope for skeletal anomalies and variations.

Statistics

A ‘litter’ was taken as the basic unit for calculations. Mean litter percentage was calculated by first calculating the percentage of affected fetuses for each abnormality in each litter and then a mean percentage was calculated across all litters for each group. A pairwise comparison was completed for each parameter between the treatment group and its control using a non-parametric method – the Kruskal–Wallis one-way analysis of variance – where P-values < 0.05 were considered to be statistically significant.

Ethical approval

The Animal Research Ethics Committee of the College of Medicine and Health Sciences, UAE University, approved the study.

Results

Maternal effects

No overt signs of maternal toxicity were discernible in any animal. In both the GD 7- and GD 13-treated animals, the average gain in weight since GD 7 of the animals in the DMSO oral group was significantly higher (GD 7 58% vs. GD 13 62%) than in the i.p. group (GD 7 54% vs. GD 13 55%) on GD 18 (P < 0.05). In the GD 13 group, both DMSO and AFB1 oral group animals were found to have gained slightly more weight than the i.p. group (DMSO 32% oral vs. 29% i.p.; AFB1 20.8% oral vs. 19.4% i.p.), though the difference was not significant. The AFB1-treated mice lost relatively more weight than DMSO-treated mice in both the GD 7 (oral 2.6% DMSO vs. 7.7% AFB1; i.p. 3.8% DMSO vs. 6.3% AFB1) and GD 13 (oral 2.5% DMSO vs. 5.8% AFB1; i.p. 2.1% DMSO vs. 5.2% AFB1) groups 1 day after treatment, but neither difference was significant. Both AFB1 and the vehicle given on GD 7 or GD 13 affected maternal food and water intake transiently; all groups registered increases in body weight during GDs 15–18.

Fetal effects

The number of fetuses in the different groups of this study was similar and not significantly reduced in most of the treatment groups. The vehicle-treated fetuses were generally heavier in weight than the AFB1-treated fetuses, but the differences were not significant, except for AFB1-treated GD 13 fetuses, which were significantly heavier than the control DMSO-treated fetuses (P < 0.05). The mean placental weight was found to vary between litters and between different treatment groups. Fetuses weighing ≥ 2 standard deviations (SDs) below the mean of the corresponding controls were considered growth restricted. The incidence of intrauterine growth restriction (IUGR) at the –2 SD level was found to be significantly higher in the GD 13 AFB1 treatment group than in the GD 7 AFB1 treatment group (Table 1 and Figure 1). These reductions in mean body weight were significant when compared with the corresponding controls (P < 0.005).

TABLE 1

Effect of AFB1 intake on different GDs on fetal implantations, resorptions and placental weights

Treated mice/fetuses/placentae GD-7 GD-13
Control fetus AFB1 fetus Control placenta AFB1 placenta Control fetus AFB1 fetus Control placenta AFB1 placenta
i.p. treated
 Number of litters 6 6 6 6 6 6 6 6
 Implantations per litter 9.17 11.67 9.17 11.67 10.17 10.17 10.17 10.17
 Fetuses, placentae per litter 8.50 10.33 8.50 10.33 9.67 9.33 9.67 9.33
 Resorptions per litter 0.67 1.33 0.67 1.33 0.50 0.83 0.50 0.83
 Fetal/placental weight (g) per litter (mean ± SD) 1.184 ± 0.07 1.157 ± 0.05 0.134 ± 0.012 0.126 ± 0.011 1.265 ± 0.134 0.927 ± 0.068**** 0.126 ± 0.019 0.111 ± 0.011
Oral treatment
 Number of litters 6 6 6 6 5 6 5 6
 Implantations per litter 9.33 10.33 9.33 10.33 9.10 10.00 10.80 10.00
 Fetuses, placentae per litter 8.50 9.67 8.50 9.67 8.50 9.33 10.20 10.20
 Resorptions per litter 0.83 0.67 0.83 0.67 0.60 0.67 0.60 0.67
 Fetal/placental weight (g) per litter (mean ± SD) 1.190 ± 0.099 1.287 ± 0.123 0.117 ± 0.006 0.130 ± 0.013 1.218 ± 0.032 1.128 ± 0.065 0.129 ± 0.009 0.117 ± 0.020

****P < 0.001.

P < 0.05 when compared with the corresponding control values.

FIGURE 1

Aflatoxin B1-induced intrauterine growth retardation and skeletal abnormalities. (a) Control fetus at the same stage as the others; (b) a growth-retarded fetus; (c) fetus with supraoccipital abnormalities (supraoccipital stage 3); and (d) fetus with lumbar abnormalities (occipitalization of the atlas). At, atlas; LR, lumbar rib; SO, supraoccipital stage 2.

HMJ-612-fig1a.jpgHMJ-612-fig1b.jpgHMJ-612-fig1c.jpgHMJ-612-fig1d.jpg

Fetal malformations

There were no treatment-related external findings, and exencephaly in a single fetus was not considered treatment related. Irrespective of the gestation stage at which treatment occurred, the DMSO-treated control fetuses did not show any discernible defects in their external morphology. However, in the GD 7 treatment group, among fetuses whose mothers had been given an i.p. or an oral dose of AFB1, 24.5% and 12%, respectively, showed reduced kidney size. In the GD 13 treatment group, the corresponding values were 23% and 20%, respectively (Table 2 and Figure 2). In the corresponding control groups all fetuses were observed to have normal-sized kidneys. Open eye anomalies were observed in three fetuses in the GD 7 AFB1 group. One fetus in the AFB1-treated group on GD 7 had a unilateral undescended testis, whereas none of the fetuses in any of the other groups had undescended testes (see Table 1).

TABLE 2

Morphological effects of AFB1 treatment in mouse fetuses on GD 7 and 13

Description of fetuses DMSO AFB1
GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral
Number of fetuses 51 51 56 50 57 58 56 55
Male (n) 29 27 39 25 29 22 26* 24
Female (n) 22 24 17 25 28 36 30* 31
IUGR (n) 0 0 0 0 10* 9* 13* 21*
Exencephaly (n) 0 0 0 0 1 0 0 0
Eyes open (n) 0 0 1 0 2 1 0 0
Kidney small size (right and left) (n) 0 0 0 0 14* 7* 13* 11*
Undescended testis (unilateral) (n) 0 0 0 0 1 0 0 0

*P < 0.05 when compared with corresponding controls.

FIGURE 2

Magnification 0.9× used for both (a) normal and (b) experimental fetus showing small left kidney and undescended testis in the fetus of AFB1-treated mice.

HMJ-612-fig2a.jpgHMJ-612-fig2b.jpg

Skeletal defects

Skull bones

Most of the vehicle-treated control fetuses were observed to have developed ossification centres appropriate for their gestational age. The exoccipital bone was adequately ossified. Most of them had their supraoccipital bones at stages 5 and 6 of development (Table 3), characterized by growth and complete fusion of the bilateral primordia. Some of the fetuses of GD 13 controls (one litter) had a non-significant decrease in size of the facial bones, discernible with the stereomicroscope. Up to 50% of fetuses of AFB1-treated i.p. mice on GD 13 had a reduced size of maxilla and mandible, whereas in the remainder progressive ossification around Meckel’s cartilage was clearly evident. A large number of other facial and basicranial bones of reduced size were also seen in both GD 7 and GD 13 AFB1-treated groups. In the corresponding DMSO controls the cranial bones were only affected in approximately 3% of fetuses. A statistically significant number of supraoccipital bones (40–60%) of the GD 7 and GD 13 AFB1-treated fetuses in the oral and i.p. groups were at developmental stages 1–4 (figure 1 of Ariyuki et al.31); in contrast, 100% of the control fetuses had advanced to stages 5 and 6. By GD 18, in most fetuses of the GD 13 AFB1-treated mothers, the majority of craniofacial bones were small and poorly ossified. For example, 100% of control fetuses had two ossicles in the middle ear but 6–13% of AFB1 fetuses had only a single ossified ossicle.

TABLE 3

Aflatoxin B1-induced facial skeletal abnormalities in mouse fetuses

Part examined DMSO, n (%) AFB1, n (%)
GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral
Number of skeletons examined 36 32 35 31 37 36 38 35
Small size
 Mandible 0 0 1 (2.9) 0 13 (35.1)*** 10 (27.8)*** 19 (50.0)*** 9 (25.7)***
 Maxilla 0 0 1 (2.9) 0 13 (35.1)*** 10 (27.8)*** 19 (50.0)*** 9 (25.7)***
 Zygomatic bone 0 0 1 (2.9) 0 2 (5.4) 1 (2.8) 4 (10.5) 2 (5.7)
 Nasal 0 0 1 (2.9) 0 2 (5.4) 1 (2.8) 4 (10.5) 2 (5.7)
 Frontal 0 0 1 (2.9) 0 11 (29.7)*** 10 (27.8)*** 16 (42.1)*** 8 (22.9)***
 Parietal 0 0 1 (2.9) 0 8 (21.6)*** 6 (16.7)* 10 (26.3)** 6 (17.1)**
 Interparietal 0 0 1 (2.9) 0 2 (5.4) 2 (5.6) 3 (7.9) 1 (2.9)
 Exoccipital 0 0 1 (2.9) 0 0 0 0 0
 Ethmoid 0 0 1 (2.9) 0 9 (24.3)*** 7 (19.4)** 11 (28.9)** 6 (17.1)**
 Presphenoid 0 0 1 (2.9) 0 9 (24.3)*** 7 (19.4)** 13 (34.2)*** 6 (17.1)**
 Basisphenoid 0 0 1 (2.9) 0 8 (21.6)*** 7 (19.4)** 13 (34.2)*** 5 (14.3)*
 Basioccipital 0 0 1 (2.9) 0 7 (18.9)** 5 (13.9)* 8 (21.1)* 5 (14.3)*
 Tympanic ring 0 0 1 (2.9) 0 7 (18.9)** 6 (16.7)* 10 (26.3)** 5 (14.3)*
 Hyoid 0 0 1 (2.9) 0 2 (5.4) 1 (2.8) 3 (7.9) 1 (2.9)
Supraoccipital
 1–4 0 0 0 0 15 (40.5)*** 15 (41.7)*** 24 (63.2)*** 14 (40.0)***
 5–6 36 (100.0) 32 (100.0) 35 (100.0) 31 (100.0) 22 (59.5)*** 21 (58.3)*** 14 (36.8)*** 21 (60.0)***
 Small size 1 (2.8) 0 0 0 5 (13.5)* 5 (13.9)* 9 (23.7)*** 4 (11.4)*
Ossicle
 0–1 0 0 0 0 3 (8.1)* 4 (11.1)* 5 (13.2)* 2 (5.7)*
 2 36 (100.0) 32 (100.0) 35(100.0) 31 (100.0) 33 (89.2) 32 (88.9) 32 (84.2)* 33 (94.3)
 3 0 0 0 0 1 (2.7) 0 1 (2.6) 0

*P < 0.05; **P < 0.005; ***P < 0.003.

Vertebrae

The vertebral column of full-term normal mouse fetuses comprises seven cervical, 13 thoracic, six lumbar, six sacral and a variable number of coccygeal vertebrae. There is a low incidence of 14 thoracic or five lumbar vertebrae. The number of ossified coccygeal vertebrae varies from six to eight in normal full-term fetuses. At this stage of development each typical vertebra consists of a centrum and two neural (vertebral) arches. Although the number of cervical centra was seven in 94–100% of control fetuses, only 47–71% of AFB1-treated group fetuses had a similar extent of ossification, with the rest having fewer ossified cervical centra; however, all fetuses had the full complement of seven cervical arches (Table 4). Caudal (tail) vertebral arches seemed minimally affected by AFB1 treatment. Although 88–97% of control fetuses had 6–9 coccygeal centra visible, a significant number of AFB1-treated fetuses exhibited a reduced number of ossified caudal centra. A significantly higher proportion of fetuses of the AFB1 group had only 0–4 coccygeal vertebral bodies. This reduction was in keeping with the increased IUGR characteristics of the AFB1 group fetuses. Fusion of thoracic vertebral arches was sporadically seen in the GD 13 AFB1 group fetuses (Figure 3).

TABLE 4

Aflatoxin B1-induced vertebral abnormalities in mouse fetuses

Skeletal abnormalities DMSO, n (%) AFB1, n (%)
GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral
Number of skeletons examined 36 32 35 31 37 36 38 35
Presacral vertebrae
 Cervical 7/Thoracic 13/Lumbar 6 35 (97.2) 32 (100.0) 34 (97.1) 30 (96.8) 34 (91.9) 33 (91.7) 32 (84.2)* 33 (94.3)
 Cervical 7/Thoracic 13/Lumbar 6 (+/–) 1 (2.8) 0 1 (2.9) 1 (3.2) 3 (8.1) 3 (8.3) 6 (15.8)* 2 (5.7)
Cervical arch (7–7) 36 (100.0) 32 (100.0) 35 (100.0) 31 (100.0) 37 (100.0) 36 (100.0) 38 (100.0) 35 (100.0)
Cervical body (7) 34 (94.4) 31 (96.9) 34 (97.1) 31 (100.0) 23 (62.2)*** 20 (55.6)*** 18 (47.4)*** 25 (71.4)
 0 1 (2.8) 0 0 0 13 (35.1)*** 16 (44.4)*** 18 (47.4)*** 10 (28.6)
 3 1 (2.8) 1 (3.1) 1 (2.9) 0 1 (2.7) 0 2 (5.3) 0
Body with decreased ossification
 2–6 0 0 0 0 7 (18.9)** 8 (22.2)** 12 (31.6)*** 6 (17.1)
 7 5 (13.9) 1 (3.1) 0 0 4 (10.8) 4 (11.1) 5 (13.2)* 3 (8.6)
Thoracic arch (13–13) 35 (97.2) 32 (100.0) 35 (100.0) 31 (100.0) 37 (100.0) 36 (100.0) 36 (94.7) 35 (100.0)
 < or > 13–13 1 (2.8) 0 0 0 0 0 1 (2.6) 0
 Fused 0 0 0 0 0 0 1 (2.6) 0
Thoracic body (13) 36 (100.0) 32 (100.0) 35 (100.0) 31 (100.0) 37 (100.0) 36 (100.0) 38 (100.0) 35 (100.0)
Lumbar arch (6) 36 (100.0) 32 (100.0) 32 (91.4) 30 (96.8) 35 (94.6) 33 (91.7) 34 (89.5) 33 (94.3)
 < or > 6 0 0 3 (8.6) 1 (3.2) 2 (5.4) 3 (8.3) 4 (10.5) 2 (5.7)
Lumbar body (6) 36 (100.0) 32 32 (91.4) 30 (96.8) 35 (94.6) 33 (91.7) 34 (89.5) 33 (94.3)
 < or > 6 0 0 3 (8.6) 1 (3.2) 2 (5.4) 3 (8.3) 4 (10.5) 2 (5.7)
Sacral arch (6) 36 (100.0) 32 (100.0) 35 (100.0) 31 (100.0) 37 (100.0) 36 (100.0) 38 (100.0) 35 (100.0)
Sacral body (6) 35 (97.2) 32 (100.0) 35 (100.0) 31 (100.0) 37 (100.0) 36 (100.0) 38 (100.0) 35 (100.0)
 < or > 6 1 (2.8) 0 0 0 0 0 0 0
Coccygeal vertebral arch
 0–4 36 (100.0) 32 (100.0) 35 (100.0) 31 (100.0) 35 (94.6) 33 (91.7) 34 (89.5) 33 (94.3)
 5–7 0 0 0 0 2 (5.4) 3 (8.3) 4 (10.5)* 2 (5.7)
Body (0–4) 4 (11.1) 1 (3.1) 4 (11.4) 1 (3.2) 22 (59.5)*** 19 (52.8)** 29 (76.3)*** 11 (31.4)
 5–9 32 (88.9) 31 (96.9) 31 (88.6) 30 (96.8) 15 (40.5)*** 17 (47.2)** 9 (23.7)*** 24 (68.6)

*P < 0.05; **P < 0.005; ***P < 0.003.

FIGURE 3

(a) Control fetus; (b) decreased ossification of caudal vertebrae (left-side rudimentary cervical rib); (c) decreased ossification of bones of the hindpaw (left-side cervical rib); (d) rib fusion; and (e) reduction in rib number and fusion of the arches and ribs.

HMJ-612-fig3a.jpgHMJ-612-fig3b.jpgHMJ-612-fig3c.jpgHMJ-612-fig3d.jpgHMJ-612-fig3e.jpg

Ribs and sternum

Variations in the frequency of lumbar and cervical ribs did not follow any discernible pattern in either the control or the experimental groups; however, the occurrence of supernumerary ribs was particularly pronounced in the fetuses of the GD 7 AFB1 treatment group (73% of the i.p.-treated group had right cervical ribs and 68% had left cervical ribs). About 44–52% of the fetuses of the other AFB1-treated groups had either left or right cervical ribs (Table 5 and Figures 24). Cervical ribs were attached dorsally to the C7 transverse processes; their ventral ends were free or found attached to the first thoracic rib or to the manubrium sterni. To be considered lumbar ribs, the occasional ribs in the lumbar region were required to be one-half or more than one-half of the length of the last thoracic rib. The majority of the control and experimental fetuses had seven sternal ribs each although a few had eight (Table 4). The eighth pair of sternal ribs was attached ventrally to the junction between sternebrae 5 and 6 and dorsally to the corresponding thoracic vertebra. There were six sternebrae in all of the control fetuses, but about 1% of fetuses of the AFB1 i.p. groups treated on GD 7 or GD 13 had more than six sternebrae. Reduction rather than an increased number of sternebrae characterized the AFB1 groups.

TABLE 5

Aflatoxin B1-induced rib abnormalities in mouse fetuses

Rib abnormality DMSO, n (%) AFB1, n (%)
GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral
Number of skeletons examined 36 32 35 31 37 36 38 35
Cervical ribs
 Left 8 (22) 6 (19) 9 (26) 4 (12.9) 25 (68)*** 17 (47)* 19 (50)* 18 (51)***
 Right 10 (28) 8 (25) 9 (26) 6 (19.4) 27 (73)*** 16 (44) 20 (53)* 17 (49)*
Thoracic ribs (13–13) 36 (100) 32 (100) 35 (100) 31 (100.0) 36 (97) 35 (97) 38 (100) 35 (100)
 < or > 13–13 0 0 0 0 1 (2.7) 1 (2.8) 0 0
 Wavy 1 (2.8) 0 0 0 1 (2.7) 0 0 0
Lumbar ribs (left) 0 0 0 0 4 (11)* 3 (8.3) 1 (2.6) 1( 2.9)
Lumbar ribs (right) 1 (2.8) 0 0 0 4 (11)* 3 (8.3) 1 (2.6) 0
Hypoplasia 0 0 0 0 1 (2.7) 2 (5.6) 2 (5.3) 0
Sternal ribs (7–7) 35 (97) 32 (100) 35 (100) 31 (100.0) 36 (97) 36 (100) 36 (95) 35 (100)
 < or > 7 1 (2.8) 0 0 0 1 (2.7) 0 2 (5.3) 0
 6 sternebrae 36 (100) 32 (100) 35 (100) 31 (100) 36 (97) 36 (100) 37 (97) 35 (100)
 < or > 6 0 0 0 0 1 (2.7) 0 1 (2.6) 0
Sternebrae
 Absent 1 (2.8) 0 0 0 2 (5.4) 1 (2.8) 3 (7.9) 2 (5.7)
 Decreased ossification 4 (11) 2 (6.3) 1 (2.9) 1 (3.2) 19 (51.4)*** 13 (36)*** 22 (58)*** 14 (40)***
 Fused or curved 0 0 0 0 1 (2.7) 0 1 (2.6) 0
 Zigzag 0 0 1 (2.9) 0 2 (5.4) 1 (2.8) 3 (7.9) 0

*P < 0.05; **P < 0.005; ***P < 0.001 compared with the corresponding control values.

FIGURE 4

Sternal anomalies. (a) Control; (b) reduced number of sternebrae; (c) reduced number of sternebrae and fusion; (d) reduced number of sternebrae and decreased ossification; (e) reduced number of sternebrae and decreased ossification; and (f) decreased ossification of sternebrae.

HMJ-612-fig4a.jpgHMJ-612-fig4b.jpgHMJ-612-fig4c.jpgHMJ-612-fig4d.jpgHMJ-612-fig4e.jpgHMJ-612-fig4f.jpg

Limb skeleton

The limb skeleton proximal to the wrist/ankle was not affected by DMSO or AFB1 treatment. However, in keeping with the overall growth restriction, limb skeletons of the AFB1 treatment group fetuses appeared smaller than those of fetuses in the control group. The small bones of the forelimbs were not the same in all groups; in about 14–19% of fetuses in the AFB1 treatment group 0–3 metacarpals were ossified, whereas in 97–100% of control group fetuses four or five metacarpals were ossified (Table 6 and Figure 5). The phalanges of the AFB1 group significantly lacked ossification compared with the control fetuses. The tarsals, metatarsals and corresponding phalanges were also significantly delayed in their ossification and growth.

TABLE 6

Aflatoxin B1-induced limb skeletal abnormalities in mouse fetuses

Limb skeleton DMSO, n (%) AFB1 treated, n (%)
GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral GD 7 i.p. GD 7 oral GD 13 i.p. GD 13 oral
Number of skeletons examined 36 32 35 31 37 36 38 35
Hand – metacarpals
 0–3 1 (2.8) 0 0 0 7 (19)* 5 (14)* 7 (18)* 5 (14)*
 4–5 35 (97) 32 (100) 35 (100) 31 (100) 30 (81) 31 (86)* 31 (82)* 30 (86)*
Hypoplasia 0 0 0 0 1 (2.7) 0 2 (5.3) 0
Phalanges
 0–5 1 (2.8) 0 0 0 6 (16) 6 (17)* 9 (24) 5 (14)*
 6–10 3 (8) 2 (6) 2 (5.7) 2 (6.5) 14 (38) 8 (22) 16 (42) 10 (29)*
 11–12 32 (89) 30 (94) 33 (94) 29 (93) 17 (46) 22 (61)*** 13 (34) 20 (57)***

*P < 0.05; **P < 0.005; ***P < 0.001.

FIGURE 5

(a) Control; and (b) observe absence and poor ossification of bones of the forepaw.

HMJ-612-fig5a.jpgHMJ-612-fig5b.jpg

Discussion

This study has clearly demonstrated the prevalence of extensive delayed skeletal ossification, IUGR and bilateral decreased kidney size in mouse fetuses exposed to AFB1 in utero via maternal i.p. or oral dosing. The toxin was dissolved in DMSO and administered to groups of mice in a single oral or i.p. dose at the early or late stages of organogenesis. No significant signs of maternal toxicity or fetal resorption were observed. Data on the reproductive toxicological effects of aflatoxins in the literature are sparse16,3237 and these studies in rabbit, rat, mouse, hamster and chick fetuses appear to be limited by inadequate information about the vehicle used, route of administration, frequency of dosing and the developmental stages at which fetuses were exposed to AFB1. For example, DiPaolo et al.16 administered 4 mg/kg i.p. aflatoxin to hamsters on GD 8 and sacrificed them on GD 9. They found that a large number (17.6%) of fetuses were dead or resorbed, and 30% were malformed; of those hamsters sacrificed on GD 12, 23% of fetuses were found to be malformed and 30% were dead or resorbed. Considering the metabolic properties of aflatoxins, one would assume that in this study pregnancy was terminated too soon and the fetuses’ repair mechanisms had too little time in utero to respond to the treatment. In the same study,16 the authors also treated C3H mice with similar doses of AFB1 and obtained entirely different results – there were no fetal malformations and no signs of the liver pathology that was present in hamsters treated with AFB1.

Feeding 10 p.p.m. aflatoxin to rats in the second half of pregnancy was reported16 to result in nodular changes in the liver in four and cholangiocarcinoma in one of 79 offspring. AFB1 is a genotoxic hepatocarcinogen that induces the formation of DNA adducts, leading to genetic changes in the target cells that cause DNA strand breakage, DNA base damage and oxidative damage and may ultimately lead to cancer. Chemical modification of the bases in DNA or amino acids in proteins leads to the formation of DNA adducts. Approximately half of human cancers are as the result of a mutation in the TP53 gene. Grice et al.33 found a variety of benign and malignant histological changes in the liver of the progeny of rats given contaminated groundnut meal containing 10 p.p.m. AFB1, whereas Yamamoto et al.34 found cleft palate, open eyelid, wavy ribs and bent long bones in the fetuses of mice treated with 32 mg/kg aflatoxin given on 2 consecutive days during the period of organogenesis. In another study, multiple doses of an aflatoxin or combinations of aflatoxins in maize oil3537 were injected into pregnant rabbits. Among the fetuses of rabbits treated with 0.1 mg/kg AFB1, there were reductions in crown to rump length and weight, enlarged eye sockets, wrist drop, agenesis of caudal vertebrae, incomplete ossification of skull bones, bent metacarpals, microphthalmia and cardiac defects. The characteristic histological findings of fetal tissues were distortion of normal hepatic cord pattern and reduced megakaryocytes in the liver, fusion of auriculoventricular valves, mild degenerative changes in myocardial fibres, microphthalmic eyes and lenticular degeneration. In a preliminary study we found that AFB1 did not form a homogeneous suspension in maize oil. Therefore, we dissolved AFB1 in DMSO and administered it in single doses at early (GD 7) or late (GD 13) stages of organogenesis. The results of our experiments indicate that the mouse fetuses at the early and late stages of organogenesis have a particular propensity to IUGR and decreased ossification of the axial and appendicular skeletal constituents.

The ability of AFB1 to cross the placenta rapidly has been shown in several studies11,13,14 and aflatoxin–albumin adducts have also been detected in both maternal and cord blood.38 In addition to forming AFB-8,9-epoxide (which is mainly responsible for the carcinogenicity of AFB1), AFB1 can be metabolized by NADPH-dependent reductase to a carcinogenic metabolite, aflatoxicol (AFL).39 AFL can be reconverted to AFB1, which can be further metabolized; AFL can itself bind to DNA and is as potent a carcinogen as AFB1.40,41 AFB1 is metabolized by CYP1A2 and 3A (3A4, 3A5, and 3A7) enzymes into several metabolites, which can react with DNA and proteins.42,43 In the adult liver, CYP1A2 and 3A4 are the main enzymes catalysing AFB1 metabolism,44,45 whereas in the human prenatal liver, CYP3A7 plays a major role in AFB1 metabolism.46 Although the human placenta may not be able to metabolize AFB1 through this route47,48 because of low levels of CYP enzymes,4749 the presence of lipoxygenase in the human term placenta and in the intrauterine conceptal tissue (including placenta) at 8–10 weeks of gestation means that it is capable of epoxidation of AFB1.50 There is evidence from placental perfusion experiments that AFB1 is rapidly transferred and is metabolized to AFL in the human placenta; because AFL is less mutagenic than, but putatively as carcinogenic as, AFB1, the formation of AFL may not protect the fetus from the toxicity of AFB1.51

Mouse fetuses of GD 7 and GD 13 correspond to Theiler stage (TS) 10–11 and TS22, respectively.52 Of particular relevance to our study are the developmental events occurring at this stage in the skeletal system as well as overall growth of the fetus. Cartilaginous primordia of the basicranium, mesenchymal condensation of the primordia of the membrane bones, digital rays of the forelimbs and segmentation of the proximal parts of the limbs are all in place by GD 13.5 (TS22), although most skeletal structures will develop their ossification centres only subsequently. The results of this study suggest that mouse fetuses of the early and late embryogenesis periods are sensitive to the noxious effects of AFB1, manifesting in delayed ossification and growth restriction. The less severe growth restriction observed in the GD 7 treatment group (compared with fetuses in the GD 13 treatment group) is possibly a reflection of repair and compensatory growth restoration mechanisms occurring over a longer period of time available between GD 7 and 18. In contrast, GD 13 treatment groups had a relatively shorter period in utero and therefore only had a limited opportunity for possible restorative growth. This assumption is supported by the results of studies in which mouse fetuses exposed to mitomycin C at primitive streak stage53 or to a low dose of gamma radiation on GD 1354,55 subsequently exhibited significant restoration of growth. The authors of the latter study refer to GD 13 as the vulnerable early fetal period. Their early fetal period is our late embryonic period in terms of Theiler’s developmental staging (TS 22–23) and GD 13–14 in a chronological sense.

Caudal vertebrae are of somite origin. The craniofacial skeleton, caudal vertebrae and bones of the forepaws and hindpaws were found to exhibit delayed ossification. This indicates that AFB1 can elicit deleterious and developmental stage-sensitive responses in this strain of mouse. The mechanism of AFB1-induced developmental disorders is not fully understood, but is probably arrested mitosis and a reduction in cell proliferation and differentiation, characteristic of the organogenesis period of development.56,57

Others have implicated maternal malnutrition in fetal defects.58 In our study there was only a transient reduction in food and water consumption and maternal body weight gain in AFB1 treatment groups. In addition to the direct embryotoxic effects of AFB1 treatment, a possible contribution of the transient reduction in food intake must be taken into consideration in interpreting IUGR. By GD 7, the placental and fetal membranes are not fully developed in mouse fetuses; in our animals that were injected with AFB1 on GD 13 the placenta was fully functional, in contrast to the placentae of mice treated on GD 7. These differences in the functional and morphological effects of treatment on the placenta may also be speculated to have a differential role in AFB1 transport and metabolism and, thus, may contribute to the differences in the frequency and severity of skeletal hypoplasia and growth restriction of the fetuses.51 In our study no increase in exencephaly in AFB1-treated animals was observed, nor were there obvious external or visceral abnormalities, as have been reported in previous studies in mice administered AFB1. Apart from the few cases of rib and vertebral arch fusion, there was no obvious skeletal malformation. However, the extensive skeletal ossification defect taken in the context of growth restriction may be regarded as a growth-inhibiting property of the toxin.

However, a significantly increased frequency of small kidneys was seen in the treatment groups. This has not been reported in previous studies in mice. A few reports have documented renal damage in rats59 and a study by Grosman et al.60 demonstrated that rats are acutely sensitive to the nephrotoxic effects of a single dose of AFB1. Mayura et al.61 demonstrated localized degeneration of distal convoluted tubules at the corticomedullary junction in the kidneys of rats animals treated with AFB1 (2 mg/kg body weight), dosed orally during GDs 6–13. Arora et al.62 reported that the kidneys of mice also excreted AFB1, and that the renal medulla was quite sensitive to this mycotoxin. It is likely that this effect on the kidneys is dose dependent in mice because of their ability to metabolize AFB1 to less toxic metabolites by conjugation to glutathione by glutathione S-transferase. This is why there are few deleterious effects on fetuses at doses (2–4 mg/kg) that are toxic to rats, hamsters and rabbits, but at higher doses, like the one administered in our experiment (20 mg/kg) and in other experiments (32 mg/kg),34 the enzyme responsible for detoxification is not able to meet the increased demand of this large dose.

Apart from hepatocellular carcinoma, other effects of AFB1 that have not been thoroughly studied in humans include the possible consequences of AFB1 exposure during pregnancy on fetal development. There are a few reports showing exposure to aflatoxins during pregnancy can cause stillbirth, low birthweight of offspring6365 and childhood cancer in genetically susceptible subgroups.10,66,67 Our experiments taken together with studies of other investigators are indicative that mice fetuses at the early and late organogenesis stages are especially susceptible to the deleterious effects of aflatoxins. This can also be true for humans and, therefore, care should be taken in how grains and nuts are stored. The fetus is particularly at risk because of its immaturity and fast-developing organs, which make it more susceptible to environmental toxins such as AFB1. Public education on this matter should be one of the priority issues for preventative health services.

Funding

The authors disclose receipt of the following financial support for the research and/or authorship of this article: Research Affairs at UAE University, under contract no. 17-7-11/02.

References

1. 

Van Egmond HP. Current situation on regulation for mycotoxins. Overview of tolerance and status of standard methods of sampling analysis. Food Addit Contam 1989; 6:139–88. http://dx.doi.org/10.1080/02652038909373773

2. 

Sun G, Wang S, Hu X, et al. Co-contamination of aflatoxin B1 and fumonisin B1 in food and human dietary exposure in three areas of China. Food Addit Contam 2011; 28:461–70. http://dx.doi.org/10.1080/19440049.2010.544678

3. 

Pietri A, Bertuzzi T, Agosti B, Donadini G. Transfer of aflatoxin B1 and fumonisin B1 from naturally contaminated raw materials to beer during an industrial brewing process. Food Addit Contam 2010; 27:1431–9. http://dx.doi.org/10.1080/19440049.2010.489912

4. 

Viegas S, Veiga L, Malta-Vacas J, et al. Occupational exposure to aflatoxin (AFB1) in poultry production. J Toxicol Environ Health 2012; 75:1330–40. http://dx.doi.org/10.1080/15287394.2012.721164

5. 

Sorenson WG, Jones W, Simpson J, Davidson JI. Aflatoxin in respirable airborne peanut dust. J Toxicol Environ Health 1984; 14:525–33. http://dx.doi.org/10.1080/15287398409530603

6. 

Selim MI, Juchems AM, Popendorf W. Assessing airborne aflatoxin B1 during on-farm grain handling activities. Am Ind Hyg Assoc J 1998; 59:252–6. http://dx.doi.org/10.1080/15428119891010514

7. 

Moss MO. Mycotoxins of Aspergillus and other filamentous fungi. J Appl Bacteriol Symp 1989; 18:S69–81.

8. 

Wu HC, Santella R. The role of aflatoxins in hepatocellular carcinoma. Hepat Mon 2012; 12:e7238. http://dx.doi.org/10.5812/hepatmon.7238

9. 

Maclean M, Dutton MF. Cellular interactions and metabolism of aflatoxins. Update Pharmacol Therap 1995; 65:163–92. http://dx.doi.org/10.1016/0163-7258(94)00054-7

10. 

Ramsdell HS, Eaton DI. Species susceptibility to aflatoxin B1 carcinogenesis: comparative kinetics of microsomal biotransformation. Cancer Res 1990; 50:615–20.

11. 

Abdulrazzaq YM, Osman N, Ibrahim A. Fetal exposure to aflatoxins in the United Arab Emirates. Ann Trop Paediatr 2002; 22:3–9. http://dx.doi.org/10.1179/027249302125000094

12. 

Abdulrazzaq YM, Osman N, Yousif ZM, Al Falahi S. Aflatoxin M1 in breast-milk of UAE women. Ann Trop Paediatr 2003; 23:173–9. http://dx.doi.org/10.1179/027249303322296484

13. 

Denning DW, Allen R, Wilkinson AP, Morgan MR. Transplacental transfer of aflatoxin in humans. Carcinogenesis 1990; 11:1033–5. http://dx.doi.org/10.1093/carcin/11.6.1033

14. 

Hsieh LL, Hsieh TT. Detection of aflatoxin B1-DNA adducts in human placenta and cord blood. Cancer Res 1993; 53:1278–80.

15. 

Cillievici O, Moldovan A, Ghidus E. Action of aflatoxin B1 on pregnant rats. Morphol Embryol 1980; 26:125–31.

16. 

DiPaolo JA, Elis J, Erwin H. Teratogenic response by hamsters, rats and mice to aflatoxin B1. Nature 1967; 215:638–9. http://dx.doi.org/10.1038/215638b0

17. 

Arora RG, Frolen H, Nilsson A. Interference of mycotoxins with prenatal development of the mouse. I. Influence of aflatoxin B1, ochratoxin A and zearolone. Acta Vet Scand 1981; 22:524–34.

18. 

Roll R, Matthiaschk G, Korte A. Embryo toxicity and mutagenicity of mycotoxins. J Environ Pathol Toxicol Oncol 1990; 10:1–7.

19. 

Sharma RA, Farmer PB. Biological relevance of adduct detection to the chemoprevention of cancer. Clin Cancer Res 2004; 10:4901–12. http://dx.doi.org/10.1158/1078-0432.CCR-04-0098

20. 

Martin J, Dufour JF. Tumor suppressor and hepatocellular carcinoma. World J Gastroenterol 2008; 14:1720–33. http://dx.doi.org/10.3748/wjg.14.1720

21. 

Jackson PE, Kuang SY, Wang JB, et al. Prospective detection of codon 249 mutations in plasma of hepatocellular carcinoma patients. Carcinogenesis 2003; 24:1657–63. http://dx.doi.org/10.1093/carcin/bgg101

22. 

Dohnal V, Wu Q, Kuca K. Metabolism of aflatoxins: key enzymes and interindividual as well as interspecies differences. Arch Toxicol 2014; 88:1635–44. http://dx.doi.org/10.1007/s00204-014-1312-9

23. 

Sun LH, Lei MY, Zhang NY, Zhao L, Krumm CS, Qi DS. Hepatotoxic effects of mycotoxin combinations in mice. Food Chem Toxicol 2014; 74:289–93 http://dx.doi.org/10.1016/j.fct.2014.10.020

24. 

Bassir O, Adekunle A. Teratogenic action of aflatoxin B1, palmotoxin B0 and palmotoxin G0 on the chick embryo. J Pathol 1970; 102:49–51. http://dx.doi.org/10.1002/path.1711020109

25. 

Wilson JG. Environment and Birth Defects. New York: Academic Press; 1973.

26. 

Bastaki SA, Osman N, Kochiyil J, Shafiullah M, Padmanabhan R, Abdulrazzaq YM. Toxicogenetics of aflatoxin in pregnant mice. Int J Toxicol 2010; 29:425–31. http://dx.doi.org/10.1177/1091581810369565

27. 

Padmanabhan R, Abdulrazzaq YM, Bastaki SM, Nurulain M, Shafiullah M. Vigabatrin (VGB) administered during late gestation lowers maternal folate concentration and causes pregnancy loss, fetal growth restriction and skeletal hypoplasia in the mouse. Reprod Toxicol 2010; 29:366–77. http://dx.doi.org/10.1016/j.reprotox.2010.02.005

28. 

Sterz H, Lehmann H. A critical comparison of the free hand razor blade dissection method according to Wilson with an in situ sectioning method for rat fetuses. Terat Carcin Mutagen 1985; 5:347–54. http://dx.doi.org/10.1002/tcm.1770050505

29. 

McLeod MJ. Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and alizarin red S. Teratology 1980; 22:299–301. http://dx.doi.org/10.1002/tera.1420220306

30. 

Inouye M. Differential staining of cartilage and bone in mouse skeleton by Alcian blue and alizarin red S. Congenital Anomalies 1976; 16:171–3.

31. 

Ariyuki F, Higaki K, Yasuda M. Staging of ossification in the supraoccipital bone in pre-term fetuses. Congenital Anomalies 1980; 20:375–81.

32. 

Geissler F, Faustman EM. Developmental toxicity of aflatoxin B1 in the rodent embryo in vitro: contribution of exogenous biotransformation systems to toxicity. Teratology 1988; 37:101–11. http://dx.doi.org/10.1002/tera.1420370203

33. 

Grice HC, Moodie CA, Smith DC. The carcinogenic potential of aflatoxin or its metabolites in rats from dams fed aflatoxin pre- and post-partum. Cancer Res 1973; 33:262–8.

34. 

Yamamoto Y, Kihara Y, Tanimura T. Effects of aflatoxin B1 on teratogenicity of mice. Teratology 1981; 24:25A.

35. 

Wangikar PB, Dwivedi P, Sinha N, Sharma AK, Telang AG. Effects of aflatoxin B1 on embryo fetal development in rabbits. Food Chem Toxicol 2005; 43:607–15. http://dx.doi.org/10.1016/j.fct.2005.01.004

36. 

Wangikar PB, Dwivedi P, Sinha N, Sharma AK, Telang AG. Teratogenic effects in rabbits of simultaneous exposure to ochratoxin A and aflatoxin B1 with special reference to microscopic effects. Toxicology 2005; 215:37–47. http://dx.doi.org/10.1016/j.tox.2005.06.022

37. 

Wangikar PB, Dwivedi P, Sinha N, Sharma AK, Telang AG. Teratogenic effects in rats of simultaneous exposure to ochratoxin A and aflatoxin B1: II. Histopathological features of teratological anomalies induced in fetuses. Birth Defects Res B 2004; 71:352–8. http://dx.doi.org/10.1002/bdrb.20022

38. 

Turner PC, Collinson AC, Cheung YB, et al. Aflatoxin exposure in utero causes growth faltering in Gambian infants. Int J Epidemiol 2007; 36:1119–25. http://dx.doi.org/10.1093/ije/dym122

39. 

Wild CP, Rasheed FN, Jawla MF, Hall AJ, Jansen LA, Montesano R. In-utero exposure to aflatoxin in west Africa. Lancet 1991; 337:1602. http://dx.doi.org/10.1016/0140-6736(91)93295-K

40. 

Wong JJ, Hsieh DP. Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proc Natl Acad Sci USA 1976; 73:2241–4. http://dx.doi.org/10.1073/pnas.73.7.2241

41. 

Bailey GS, Dashwood R, Loveland PM, Pereira C, Hendricks JD. Molecular dosimetry in fish: quantitative target organ DNA adduction and hepatocarcinogenicity for four aflatoxins by two exposure routes in rainbow trout. Mutat Res 1998; 399:233–44. http://dx.doi.org/10.1016/S0027-5107(97)00258-3

42. 

Bailey GS, Loveland PM, Pereira C, Pierce D, Hendricks JD, Groopman JD. Quantitative carcinogenesis and dosimetry in rainbow trout for aflatoxin B1 and aflatoxicol, two aflatoxins that form the same DNA adduct. Mutat Res1994; 313:25–38. http://dx.doi.org/10.1016/0165-1161(94)90030-2

43. 

Eaton DL, Gallagher EP. Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 1994; 34:135–72. http://dx.doi.org/10.1146/annurev.pa.34.040194.001031

44. 

Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H, Langouet S. Activation and detoxication of aflatoxin B1. Mutat Res 1998; 402:121–8. http://dx.doi.org/10.1016/S0027-5107(97)00289-3

45. 

Gallagher EP, Wienkers LC, Stapleton PL, Kunze KL, Eaton DL. Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res 1994; 54:101–8.

46. 

Doi AM, Patterson PE, Gallagher EP. Variability in aflatoxin B(1)-macromolecular binding and relationship to biotransformation enzyme expression in human prenatal and adult liver. Toxicol Appl Pharmacol 2002; 181:48–59. http://dx.doi.org/10.1006/taap.2002.9399

47. 

Hakkola J, Pasanen M, Hukkanen J, et al. Expression of xenobiotic-metabolizing cytochrome P450 forms in human full-term placenta. Biochem Pharmacol 1996; 51:403–11. http://dx.doi.org/10.1016/0006-2952(95)02184-1

48. 

Hakkola J, Raunio H, Purkunen R, et al. Detection of cytochrome P450 gene expression in human placenta in first trimester of pregnancy. Biochem Pharmacol 1996; 52:379–83. http://dx.doi.org/10.1016/0006-2952(96)00216-X

49. 

Pavek P, Dvorak Z. Xenobiotic-induced transcriptional regulation of xenobiotic metabolizing enzymes of the cytochrome P450 superfamily in human extrahepatic tissues. Curr Drug Metab 2008; 9:129–43. http://dx.doi.org/10.2174/138920008783571774

50. 

Datta K, Kulkarni AP. Oxidative metabolism of aflatoxin B1 by lipoxygenase purified from human term placenta and intrauterine conceptal tissues. Teratology 1994; 50:311–17. http://dx.doi.org/10.1002/tera.1420500406

51. 

Partanen HA, El-Nezami HS, Leppänen JM, Myllynen PK, Woodhouse HJ, Vähäkangas KH. Aflatoxin B1 transfer and metabolism in human placenta. Toxicol Sci 2010; 113:216–25. http://dx.doi.org/10.1093/toxsci/kfp257

52. 

Kaufman MH, Bard JBL. The Anatomical Basis of Mouse Development. San Diego: Academic Press; 1999.

53. 

Snow MH, Tam PP. Is compensatory growth a complicating factor in mouse teratology? Nature 1979; 279:555–7. http://dx.doi.org/10.1038/279555a0

54. 

Devi PU, Baskar R, Hande MP. Effect of exposure to low-dose gamma radiation during late organogenesis in the mouse fetus. Radiat Res 1994; 138:133–8. http://dx.doi.org/10.2307/3578857

55. 

Devi PU, Hossain M. Effect of early fetal irradiation on the postnatal development of mouse. Teratology 2001; 64:45–50. http://dx.doi.org/10.1002/tera.1046

56. 

Irvin TR, Poorniiyer ALS, Stevens EK, Martin JE. Parental toxicity of aflatoxins: application of mammalian cell culture systems to characterize the developmental toxicity of aflatoxins mycotoxins. Cancer Health 1991; 167:82.

57. 

Joshi MS, Joshi MV. Effect of aflatoxin B1 on early embryonic stages of the chick Gallus domesticus cultured in vitro. Indian J Exp Biol 1981; 19:528–31.

58. 

Butler WH. The effect of maternal liver injury and dietary reduction on fetal growth in the rat. Food Cosmet Toxicol 1971; 9:57–63. http://dx.doi.org/10.1016/S0015-6264(71)80116-5

59. 

Ikegwuonu FL, Egbunike GN, Emerole GO, Airc TA. The effects of aflatoxin B, on some testicular and kidney enzyme activity in rat. Toxicology 1980; 1:9–16. http://dx.doi.org/10.1016/0300-483X(80)90022-0

60. 

Grosman ME, Elias MM, Comin EJ, Rodriguez Garay EA. Alterations in renal function induced by aflatoxin B, in the rat. Toxicol Appl Pharmacol 1983; 69:319–25. http://dx.doi.org/10.1016/0041-008X(83)90255-7

61. 

Mayura K, Abdel-Wahhab MA, McKenzie KS, et al. Prevention of maternal and developmental toxicity in rats via dietary inclusion of common aflatoxin sorbents: potential for hidden risks. Toxicol Sci 1998; 41:175–82. http://dx.doi.org/10.1093/toxsci/41.2.175

62. 

Arora RG, Appelgren LE, Bergman A. Distribution of [l4C]-labeled aflatoxin B, in mice. Ada Pharmacol Toxicol 1978; 43:273–9.

63. 

De Vries HR, Maxwell SM, Hendrickse RG. Foetal and neonatal exposure to aflatoxins. Acta Pediatr Scand 1989; 787:373–8. http://dx.doi.org/10.1111/j.1651-2227.1989.tb11095.x

64. 

Abdulrazzaq YM, Osman N, Yousif ZM, Trad O. Morbidity in neonates of mothers who have ingested aflatoxins. Ann Trop Pediatr 2004; 24:145–51. http://dx.doi.org/10.1179/027249304225013420

65. 

Shuaib FM, Jolly PE, Ehiri JE, et al. Association between birth outcomes and aflatoxin B1 biomarker blood levels in pregnant women in Kumasi, Ghana. Trop Med Int Health 2010; 15:160–7. http://dx.doi.org/10.1111/j.1365-3156.2009.02435.x

66. 

IARC. Cancer in Africa: epidemiology and prevention. IARC Sci Publ 2003; 153:381–96.

67. 

Godschalk RW, Kleinjans JC. Characterization of the exposure–disease continuum in neonates of mothers exposed to carcinogens during pregnancy. Basic Clin Pharmacol Toxicol 2008; 102:109–17. http://dx.doi.org/10.1111/j.1742-7843.2007.00174.x


Comments on this article



View all comments  |  Add comment 





Home  Editorial Board  Search  Current Issue  Archive Issues  Announcements  Aims & Scope  About the Journal  How to Submit  Contact Us
Find out how to become a part of the HMJ  |   CLICK HERE >>
© Copyright 2012 - 2013 HMJ - HAMDAN Medical Journal. All Rights Reserved         Website Developed By Cedar Solutions INDIA