Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring

BACKGROUND: Chromium(III) picolinate, [Cr(pic)(3)], is a widely marketed dietary supplement. However, Cr(pic)(3) has been associated with oxidative damage to DNA in rats and mutations and DNA fragmentation in cell cultures. In isolated case reports, Cr(pic)(3) supplementation has been said to cause adverse effects, such as anemia, renal failure, liver dysfunction, and neuronal impairment. To date, no studies have been published regarding the safety of chromium picolinate supplementation to a developing fetus, although Cr(pic)(3) has been recommended for pregnant women who are diagnosed with gestational diabetes. METHODS: From gestation days (GD) 6-17, pregnant CD-1 mice were fed diets containing either 200 mg/kg Cr(pic)(3), 200 mg/kg CrCl(3), 174 mg/kg picolinic acid, or the diet only to determine if Cr(pic)(3), CrCl(3), or picolinic acid could cause developmental toxicity. Dams were sacrificed on GD 17, and their litters were examined for adverse effects. RESULTS: The incidence of bifurcated cervical arches was significantly increased in fetuses from the Cr(pic)(3) group as compared to the diet-only group. Fetuses in the picolinic acid-treated group had an incidence double that of the control group; however, this increase was not statistically significant. Fetuses in the CrCl(3) group did not differ from the controls in any variable examined. No maternal toxicity was observed in any of the treatment groups. CONCLUSIONS: High maternal oral exposures to chromium picolinate can cause morphological defects in developing offspring of mice.     

Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages

The induction of congenital malformations among the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages has been studied in two experiments. Firstly, animals were exposed to varying doses (108–504 cGy) of X-rays and mated at various time intervals (1–7, 8–14, 15–21 and 64–80 days post-irradiation), so as to sample spermatozoa, spermatids and spermatogonial stem cells. In the second experiment, only treated spermatogonial stem cells were sampled. One group of males was given a single 500-cGy dose, a second group a fractionated dose (500 + 500 cGy, 24 h apart) and a third group was left unexposed.In the first experiment, induced post-implantation dominant lethality increased with dose, and was highest in week 3, in line with the known greater radiosensitivity of the early spermatid stage. Preimplantation loss also increased with dose and was highest in week 3. There was no clear induction of either pre-implantation or post-implantation loss at spermatogonial stem cell stages.There was a clear induction of congenital malformations at post-meiotic stages, the overall incidence being 2.0 ± 0.32% in the irradiated series and 0.24 ± 0.17% among the controls. The induction was statistically significant at each dose. At the two highest doses the early spermatids (15–21 days) appeared more sensitive than spermatozoa, and at this stage the incidence of malformations increased with dose. The data from Expt. 1 on the induction of malformations by irradiation of spermatogonial stages were equivocal. In contrast, Expt. 2 showed a statistically significant induction of malformations at both dose levels (2.2 ± 0.46% after 500 cGy and 3.1 ± 0.57% after 500 + 500 cGy). The relative sensitivities of male stem cells, post-neiotic stages and mature oocytes to the induction of congenital malformations were reasonably similar to their sensitivities for specific-locus mutations, except that the expected enhancing effect of the fractionation regime used was not seen.Dwarfism and exencephaly were the two most commonly observed malformations in all series.     

Congenital defects in the offspring of male mice treated with ethylnitrosourea

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in mg/kg by treating spermatogonial stem cells was estimated to be 1.0 X 10(-4), which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.     

Dietary protein deficiency in pregnant mice and offspring

Previous studies suggest an association between dermal contact hypersensitivity and preterm delivery. We hypothesized that dietary protein deficiency produces cell-mediated immune hypersensitivity in pregnant animals and their offspring akin to those known to produce tissue damage. We compared the effects of feeding a 20% protein diet (controls) to those of feeding a 10% protein (deficient) diet ad libitum to pregnant BALB/c mice. We measured dermal contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) by the increment in ear skin thickness (swelling) 72 h after immunization and parity by the number of viable pups delivered. Dams fed the protein-deficient diet ingested less food, gained less weight and delivered fewer viable pups than the dams fed the control diet. Greater DNFB-stimulated increment in ear skin thickness was found in the protein-deficient mothers and in their offspring than in the control mothers and their offspring. We conclude that dietary protein deficiency limits parity and induces immune hypersensitivity. These findings suggest the potential for dietary protein deficiency to activate a T-cell-mediated branch of the immune response that may put pregnant animals at risk for preterm delivery.     

Effects of ATP on the stability of enzymes against heat treatment in 6-aminonicotinamide treated quail

The stabilities of liver and pectoral muscle enzymes in 6-aminonicotinamide (6-AN) treated quail against heat treatment in the presence and absence of added ATP were investigated. Only ATP level in the brain and pectoral muscle of 6-AN treated group was significantly reduced compared to the control group whereas ADP and AMP levels were not affected. In the thermal stability (55 degrees C) of liver enzymes, the activity of acetylcholinesterase (AChE) was not affected whereas the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were significantly lowered (P<0.01). The addition of 1mM ATP to liver enzyme extracts of 6-AN group afforded 4- and 1.7-fold more protection for GAPDH and LDH, respectively (P<0.01). In liver, LDH appeared to be more protected by ATP than GAPDH. In muscle, however, GAPDH and AChE activity were significantly affected but not LDH. The addition of 1mM ATP to muscle enzyme extracts of 6-AN group afforded 1.7-fold more protection for GAPDH (P<0.01) but rather inactivated AChE. A marked reduction in ATP levels in muscle did not affect specifically muscle enzyme activities only since liver enzyme activities were also affected to the same degree as muscle.     

Crowding pregnant mice affects attack and threat behavior of male offspring

Attack and threat behavior of adult male offspring of female mice crowded during the final third of pregnancy was investigated. In 5-min test pairings with an anosmic "standard opponent" which had 50 microliter of male mouse urine applied to its fur, the prenatally stressed group of males showed significantly less attack behavior; attack latency was longer and number of attacks, bites, amount of time spent attacking, and composite aggression scores were all lower, compared with the control group. Similarly, less threat behavior was observed in offspring from crowded dams; there were lower frequencies of tail rattles, rough grooms, and upright threats. Additionally, proportionally fewer males in the prenatally stressed group attacked or displayed threats. A second experiment was designed to investigate the effects of exogenous androgen on the aggressiveness of males from crowded mice: testosterone propionate administration (500 micrograms/animal/day, for 5 days prior to testing) abolished differences both in the proportion of males from crowded mice that fought and also apparently abolished differences in intensities of attack and threat behavior between groups. However, trends toward reduced aggression in prenatally crowded males remained. More detailed analysis of these responses, based only on animals that displayed aggression, revealed significantly reduced intensity of aggression in offspring from females crowded during pregnancy, indicating that testosterone propionate therapy did not completely restore this behavior. In order to reduce postnatal effects due to possible differences in mothering, all offspring were fostered to untreated mothers at birth. The results are discussed in terms of in utero exposure of male fetuses of crowded dams to stress-liberated adrenal steroids of maternal origin, and the possible consequences for the endocrine integrity of these offspring.     

Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea

ICR strain male mice injected intraperitoneally with daily doses of MNU (5–25 mg/kg) for 5 days and mated to untreated virgin females of the same strain on days 1–7, 8–14, 15–21 and 64–80 after the last dose. Copulations during these periods involve, respectively, spermatozoa, late spermatids, early spermatids, and spermatogonial stem cells at the time of the last treatment. The uterine contents were examined on day 18 of pregnancy for post-implantation losses (dominant lethality). Fetuses were examined for exteranal and skeletal abnormalities. In contrast to the results reproted for specific-locus mutations, MNU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of congenital defects and of dominant lethals over the control levels. The relative sensitivity of germ cells sampled on days 1–7, 8–21 and 64–80 to MNU-induced congenital defects was 1:1.6:2. For the induction of dominant lethals, the sensitivity ratio was 1:1.8:0.5. It is proposed that congenital defects in the offspring of mice following paternal treatment with MNU may represent mostly chromasomal rather than genic changes. Cleft palate was the most frequent of the external abnormalities, which were significantly induced in every treatment series; fused ribs were the most frequent of the skeletal abnormalities, which were significatly induced in the treatment series for spermatogonial stem cells.     

Neurobehavioral protective properties of curcumin against the mercury chloride treated mice offspring

In the present investigation, the effects of mercuric chloride (HgCl₂) on the neurobehavioural and neurochemical disruption in mice offspring was studied. A total of thirty pregnant mice were divided into six groups. Group II and III were received 150 and 300 ppm of curcumin respectively. Group IV was given 10 ppm of HgCl₂. Group V and VI were given 10 ppm of HgCl₂ with 150 and 300 ppm of curcumin respectively. In this study, treatment started from day one of pregnancy and continued until post-natal day 15 (PD 15). During weaning period, three pups in each experimental group were marked and were subjected to behavioral, physical and biochemical tests. The results revealed decreased body weight, delayed hair growth and eye opening. HgCl₂ treated pups taken more time in righting, rotating reflexes to return to normal placement, cliff avoidance compared to that of control group. HgCl₂ exposed pups showed memory and learning deficits. Anxiety behavior in treating pups was increased. Biochemical investigations showed decreased level of dopamine (DA), serotonin (5-HT) and acetylcholinesterase (AChE) in forebrain of treated pups compared to the control and curcumin groups. The protective effect of curcumin doses were significant compared to HgCl₂ group. The results indicated that the administration of curcumin showed effective activity towards biochemical and behavioral disorders obtained with the HgCl₂ treated animals. Overall, the curcumin administration revealed increased cognetion and anxiety behaviors in the treated animals. Conclusively, curcumin has a good benefits for health which can use to avoid toxicants such as Hg and other heavy metals.     

An examination of respiratory distress and chromosomal abnormalities in the offspring of male mice treated with ethylnitrosourea

A functional defect (respiratory distress), in addition to morphological defects, was induced in the offspring of male ICR mice treated with ethylnitrosourea (ENU) before mating. ENU (100 and 50 micrograms/g) was injected intraperitoneally into adult male ICR mice that were then mated with untreated females. After the cesarian operation on the 18th day of gestation, fetuses were resuscitated. In the apneic fetuses showing respiratory distress, the lung was collapsed and the ductus arteriosus was not closed. The incidence of fetuses showing respiratory distress was significantly increased with the high dose (100 micrograms/g) of ENU, and it was higher after spermatogonial exposure than after postmeiotic exposure. There was no linearity in the dose-response relationship at the lower dose (50 micrograms/g), as was the case with the specific-locus mutation. The frequency per microgram ENU of fetuses showing respiratory distress was 3.7 X 10(-4) for spermatogonial treatment (calculated at a dose of 100 micrograms/g), the value being about 10-20 times higher than that of ordinary mutations in mice. About half of the fetuses showing respiratory distress often had specific anomalies (dwarfism and gigantic thymus), but the remainder showed no morphological changes. Spermatogonial treatment produced a zero or very low incidence of translocations in the meiotic configurations of primary spermatocytes. G-band analysis of the affected F1 fetuses also revealed no visible chromosomal abnormalities (there could be small deletions or inversions) except that trisomy 19 was found in a dwarf fetus.     

Production of axial skeletal malformations with the nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in the mouse

BACKGROUND: To test whether the differentiating embryo is susceptible to the teratogenic effects of the nitric oxide (NO) synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME). METHODS: ICR-(CD-1) mice received a single intraperitoneal injection of L-NAME at 90, 150, or 300 mg/kg on Gestation Day (GD) 8 or 9. Controls were treated with vehicle on GD 8 and 9. Teratological assessments were carried out near term (GD 18). RESULTS: Maternal treatment with a single dose of L-NAME at 150 or 300 mg/kg on either GD 8 or 9 produced axial skeletal defects in the ICR (CD-1) mouse fetuses. Other treatment-related effects included increased embryo lethality and fetal growth restriction. CONCLUSIONS: This study provides evidence that in utero exposure to L-NAME can affect organogenesis of the axial skeleton.     

Ectopic expression of Hox-2.3 induces craniofacial and skeletal malformations in transgenic mice.

To better understand the role of the Hox-2.3 murine homeobox gene during development, a dominant gain-of-function mutation was generated. The developmental malformations that resulted when the chicken beta-actin promoter was used to direct widespread expression of the Hox-2.3 gene in transgenic mice included early postnatal death as well as craniofacial abnormalities, including open eyes and cleft palate. Ventricular septal defects were also observed in the hearts of three transgenic mice. Skeletal malformations were seen in the bones of the craniocervical transition, with the occipital, basisphenoid, and atlas bones deficient or misshapen. Interestingly, one mutant exhibited an extra pair of ribs as well as alterations in cervical vertebrae identities. Some of the malformations observed in Hox-2.3 gain-of-function mutants overlap with those seen in Hox-1.1 and Hox-2.2 misexpression mutants which suggests functional similarities between paralogous homeobox genes. The results of these experiments are consistent with a role for Hox-2.3 in specifying positional information during development.     

Mammary gland differentiation in hypophysectomized, pregnant mice treated with corticosterone and thyroxine.

Swiss Webster mice were hypophysectomized or sham-operated on Day 11 of pregnancy. The animals were fitted s.c. with osmotic minipumps containing either corticosterone (B) dissolved in Molecusol (Pharmatec, Alachua, FL) or the vehicle alone immediately after they were hypophysectomized. Animals in some of the experimental groups also received thyroxine (T4) in their drinking water. The mice were killed on Day 18 of gestation, and mammary tissue was homogenized and extracted for assessment of DNA, RNA, alpha-lactalbumin, and alpha-casein. Serum was assayed for placental lactogen-I (PL-I), and placental lactogen-II (PL-II), B, and T4. The concentration of PL-II in serum was elevated in the hypophysectomized mice, whereas the PL-I concentration did not differ among experimental groups. Hypophysectomy decreased both T4 and B concentrations in serum, and administration of these hormones restored their serum concentrations to normal or, in some cases, somewhat higher than normal levels. Hypophysectomy reduced the total RNA content and RNA/DNA ratio of the mammary gland, but treatment with B alone or with B and T4 restored RNA levels to those of sham-operated animals. T4 alone was ineffective in restoring RNA levels. Sham-operated animals that received hormonal treatment (B and T4) had the highest levels of RNA in the mammary tissue. Hypophysectomized animals had reduced content and concentration of alpha-lactalbumin in the mammary gland as compared to all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of Trichinella spiralis infection of pregnant mice on the future behavior of their offspring

Trichinella spiralis infections in pregnant mice had both pre- and post-natal effects on the future behavior of the offspring. Young, born of infected and reared by uninfected mothers as well as young born of uninfected and reared by infected mothers, were behaviorally distinguishable from controls that were born and reared by uninfected mothers when assessed by their tendency to emerge from their home cages into an unfamiliar open-field arena. The pre- and post-natal effects appeared to be additive, since offspring born and reared by infected mothers were most severely affected.