Specific teratogenic action of 2,4,5-trichlorophenoxyacetic acid on mouse and rat whole-embryo cultures

The effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were studied at concentrations ranging from 0.17 to 3.52 m on post-implanted mouse and rat embryos cultured for 48 hr, either in the absence of any metabolic activation system or in the presence of mouse and rat S-9 mix. 2,4,5-T proved to be a potential teratogen, at 0.88 and 1.76 m , on mouse embryos in the absence of any metabolic activation system. In the presence of mouse S-9 mix, 2,4,5-T showed a high teratogenic potential at 0.17, 0.88 and 1.76 m . In contrast, in the presence of rat S-9 mix, 2,4,5-T induced structural defects only at 1.76 m . At 3.52 m , 2,4,5-T was 100% embryolethal with or without S-9 mix. On rat embryos, 2,4,5-T was potentially teratogenic only in the presence of mouse S-9 mix, causing a significant increase in dysmorphogenic effects at 0.17, 0.88 and 1.76 m . With or without rat S-9 mix, 2,4,5,-T was only embryolethal to rat embryos at 1.76 and 3.52 m . The abnormalities mainly involved the forebrain, the midbrain and the branchial arches. These results are consistent with the known in vivo embryotoxic action of this compound.     

The teratogenicity of methoxyacetic acid in the rat

Intraperitoneal (i.p.) administration of methoxyacetic acid (MAA) to rats on Day 8, 10, 12 or 14 of pregnancy was embryolethal and teratogenic. Skeletal anomalies, hydrocephalus and dilatation of the kidney pelvis were the most common malformations. Embryonic response to MAA varied with gestational age and with dosage (0.1 to 2.5 mmol/kg). These actions are similar to those previously reported for 2-methoxyethanol (ME) and dimethoxyethyl phthalate (DMEP). Embryos were also examined on Day 12, 48 h following i.p. administration of 2.5 mmol/kg MAA. Abnormalities were comparable to those previously observed following MAA treatment of rat conceptuses in culture. These data support the conclusion that MAA is the proximal teratogenic metabolite of ME and DMEP.     

Assessment of the teratogenic potential of acrolein and cyclophosphamide in a rat embryo culture system

Rat conceptuses were explanted from the uterus on day 10.5 of gestation. The embryos within the yolk-sacs were then transferred to culture bottles containing pure male rat serum either with or without liver microsomes and NADPH. Acrolein (AC), present worldwide in the environment and one of the intermediate metabolites of cyclophosphamide (CPA), was added to these culture mediums. The conceptuses were grown for a period of 48 h after which the morphological features and their degree of differentiation were examined and the DNA and protein contents determined. The effects produced by AC were compared with those obtained by CPA treatment, using the same culture conditions. AC treated embryos and yolk-sacs showed slight but statistically significant inhibition of growth at concentrations of 100 μM and 150 μM. Higher dose levels (200 μM and 250 μM) resulted in a drastic inhibition of growth and differentiation. However, no gross structural defects were observed at the dose-levels used. In contrast, conceptuses cultivated in the presence of CPA (350 μM), liver microsomes and NADPH showed characteristic morphologic lesions. Our findings indicate that AC is lethal to embryos within a narrow dose-range, but has no teratogenic potential. Therefore, AC is not the metabolite which is responsible for the teratogenic effects observed after CPA treatment in vivo. The results also demonstrate that the postimplantation embryo culture system can discriminate between embryolethal and teratogenic effects and that whole embryos in culture can respond to teratogens in a manner similar to embryos exposed in vivo.     

Reproductive toxicological investigations with sennosides

These trials were carried out to ascertain whether sennosides have any influence on reproduction in rats and rabbits. After intragastric administration there was no evidence of any embryolethal, teratogenic or fetotoxic action. Furthermore, sennosides had no effect on the postnatal development of young animals, on the rearing behaviour of mother animals or on male or female fertility.     

In vitro embryotoxicity and teratogenic potential of etretinate and etretin

The embryotoxicity and teratogenic potential of etretinate and its main metabolite, etretin, with or without enzymatic drug activation, were investigated in vitro using the rat whole embryo culture system. The metabolizing system used was either a rat liver homogenate (S-9 mix) or esterase (carboxylic-ester hydrolase). Groups of 8–14 rat embryos (explanted on day 9.5 of pregnancy) were cultured for 48 hr with 0, 3, 10, 30 or 100 μg etretinate/ml with or without S-9 mix, or with 0, 0.03, 0.1, 0.3, 1, 3 or 10 μg etretinate/ml in the presence of 0.5 or 1 U esterase/culture flask. Yolk-sac diameters and vascularization and crown-rump and head lengths were unaffected by etretinate treatment alone but somite numbers and morphological scores were reduced at 30 and 100 μg etretinate/ml. The number of embryos with morphological anomalies was increased in the presence of 10 μg etretinate/ml but not significantly so. At higher concentrations the incidence of embryos with anomalies was 100%. Activation of etretinate in the presence of S-9 mix led to increased teratogenic activity of the test compound in vitro, although embryonic crown-rump and head lengths were comparable to control measurements up to the highest concentrations tested. When it was co-incubated with esterase, etretinate became 100 times more active, and its effects occurred in the same concentration range as the embryotoxic effects of etretin added to the culture directly. Without activation etretinate was approximately 100 times less active than etretin in producing general embryotoxicity as well as anomalies in the in vitro culture. The anomalies found after in vitro exposure of rat embryos to etretinate or etretin in this study resembled to a great extent the malformations found in vivo in several species including man. The pattern of anomalies was specific for the compounds used and was similar to that induced by other retinoids. The concentrations of etretin eliciting teratogenic effects in vitro were in the range of etretin peak plasma levels during etretinate therapy in man. Our results indicate that the whole-embryo culture system is a sensitive and valuable model for assessing the embryotoxic and teratogenic potential of retinoids in vitro.     

A model combining the whole embryo culture with human liver S-9 fraction for human teratogenic prediction

The whole embryo culture system has proved particularly useful in evaluating the role of biotransformation in dysmorphogenic processes. Using the traditional method, information on the teratogenic potential of chemicals can be obtained, but the results are difficult to extrapolate to humans. In this study, a human liver S-9 fraction was used as an enzyme source for the bioactivation of cyclophosphamide (CP) in vitro, to assess whether this model mimics more nearly the human situation. CP is a well known rodent teratogen but probably is not teratogenic in humans. The effects were compared of Aroclor-1254-induced rat liver S-9, non-induced rat liver S-9 and human liver S-9 fractions on CP teratogenicity in vitro. CP (30 μg/ml) with non-induced rat S-9 (50 μl) did not cause a significant increase in adverse effects (46.7%), whereas 100% dysmorphogenic effects were shown with induced rat S-9 (30 μl, 50 μl). CP (30–150 μg/ml) did not produce dysmorphogenesis (below 35.5%) in the presence of human S-9 (50 μl).     

Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro.

Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 micromol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.     

Mutagenicity testing of selected analgesics in Ames Salmonella strains

Acetaminophen (APAP), aspirin (ASA), phenacetin (PA) and ibuprofen (IB) were tested for mutagenic activity in the Ames Salmonella plate incorporation assay using strains TA98, TA100, TA1535, TA1537 and TA1538. These analgesics were tested in four separate tests: without metabolic activation, and in the presence of a rat, hamster or mouse liver post-mitochondrial supernatant (S-9, Aroclor 1254-induced). Treatment of all five strains of Salmonella with APAP, ASA or IB under all four metabolic conditions did not induce any appreciable increases in revertant colony counts, as compared to the negative controls. A dose-related increase in revertant colony counts, reaching levels twice the negative control values, were seen with PA at doses greater than or equal to 500 micrograms per plate. This response was only seen in strain TA100 in the presence of hamster S-9. Therefore, these findings constitute a positive result for PA in the Ames test. APAP, ASA and IB did not show any mutagenic potential under these conditions of testing. These findings are discussed along with previously published results concerning the genotoxicity of these analgesics.     

Monitoring of organ formation in rat embryos after in vitro exposure to azathioprine, mercaptopurine, methotrexate or cyclosporin A

Rat embryos in the organ formation phase (days 9.5-11.5 post coitum) were cultivated in pure rat serum in the presence of an Aroclor 1254 pretreated liver microsomal preparation (S9-mix). Various concentrations of the immunosuppressive drugs azathioprine (AZ), 6-mercaptopurine (MP), methotrexate (MTX) or cyclosporin A (CS-A) were added at the beginning of the culture period. Forty-eight hours later, malformations were observed in the AZ, MP and MTX treated embryos at concentrations as low as 1 microgram/ml, 1.8 micrograms/ml and 0.05 microgram/ml, respectively. This indicates that these drugs have a direct effect on embryonic development. They selectively affected the rhombencephalic and telencephalic brain regions. Other malformations were seen in the caudal trunk, the heart and forelimb regions, and in the vesicular structures. It is suggested that the similarity of the pharmacological action of these drugs, that is, the DNA de novo synthesis inhibition, was the cause of the comparable types of malformations observed. Higher AZ, MP and MTX concentrations caused concentration-dependent increases in the types and incidences of malformations, as well as inhibited overall growth and differentiation. CS-A, a new type of immunosuppressant agent, had no effect on the morphogenetic events at the concentrations tested. These results are generally in agreement with the literature data, indicating that AZ, MP and MTX induce malformations in whole-animal systems, whereas CY-A does not. When AZ and MTX were assayed in the rat species in vivo, on the other hand, embryolethalities and retardations, but few malformations, were observed. The possibility of controlled exposure in vitro may, therefore, offer the advantage that clearer distinctions between embryolethal and teratogenic effects can be made.     

Use of human liver S9 in the Ames test: assay of three procarcinogens using human S9 derived from multiple donors

The purpose of the present study was to examine the inter-individual variation in the mutagenicity of chemicals using a variety of human S9 fractions. For this purpose, three procarcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), benzo[a]pyrene (BP), and dimethylnitrosamine (DMN), were selected for the Ames test and their mutagenicity was examined using human liver S9 fractions prepared from 18 different donors and one pooled liver S9 fraction prepared from 15 different donors. In addition, rat S9 fraction prepared from male rats pretreated with phenobarbital and 5,6-benzoflavone (PB/BF) was used as reference in order to examine the mutagenic differences between human and rat (PB/BF) S9 fractions. The data demonstrate a large inter-individual diversity in the mutagenic response to procarcinogens. The mutagenicity of IQ and BP in the presence of a human liver S9 fraction (lot HLS-014) was equal to that observed in the presence of rat (PB/BF) S9 fraction. The mutagenicity of IQ and BP in the presence of a pooled human liver S9 fraction was lower (90 and 95%, respectively) than that observed in the presence of rat (PB/BF) S9. On the contrary, the mutagenicity of DMN in the presence of either a selected human liver S9 fraction (lot HLS-014) or pooled fraction was 8-fold higher than that found in the presence of rat (PB/BF) S9 fraction. Human liver S9 fraction (lot HLS-014) had one of the highest cytochrome P450 enzyme activities among the 18 different donors and higher than the pooled human liver S9 fraction. These results suggest that the use of both selected human liver S9 fractions with high metabolic activity (e.g., lot HLS-014 as used in this study) and a pooled S9 fraction with moderate metabolic activity could be used as a means to evaluate the inter-individual variability in mutagenic response to chemicals and to confirm positive responses from studies completed with rodent S9.     

Embryolethality of new herbicides is not detected by the micromass teratogen tests

New herbicidal compounds (11 pyrimidine-diones, 3 benzoates and 1 sulfonamide) were found to be embryolethal but not teratogenic in rats. The range of the embryolethal dose varied from 0.2 to >200 mg/kg. This broad range enabled us to validate whether proposed in vitro teratogen tests can detect the embryolethality of these herbicides. The IC₅₀ values (inhibition concentration 50%) for both differentiation and proliferation of midbrain and limb bud cells of rat embryos were determined and found to be above 50 g/ml in all cases, confirming that the herbicides were not teratogenic. No correlation, however, was observed between the embryolethality in vivo and the activities in these cells. In order to test whether the potential to cause embryolethality could be predicted and detected as a general cytotoxic effect, the inhibition of colony forming ability in V79 cells was determined. The results indicated that cytotoxicity in V79 cells may be useful for preliminary testing of the embryolethal effect of herbicides.     

Mutagenicity study of gadobenate dimeglumine formulation (E7155) (2)--Chromosome aberration test with human lymphocytes in culture

The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the chromosome aberration test in cultured human lymphocytes. Human lymphocytes were exposed to E7155 at 0.078-10 mM both in the presence and absence of S9 mix derived from rat livers. Three dose levels (2.5-10 mM) were selected for the metaphase analysis. E7155 induced no increase in the incidence of aberrant cells or polyploid cells in any treatments both in the presence and absence of metabolic activation. Thus, it is concluded that E7155 has shown no evidence of clastogenic or polyploidy-inducing activity under these experimental conditions.     

Effect of oral dosing vehicles on the developmental toxicity of flubendazole in rats

Flubendazole was suspended in deionized water or olive oil and administered by gavage once daily to pregnant rats on Days 8-15 of pregnancy to examine if the embryolethal and teratogenic doses were affected by the vehicles used. Flubendazole in olive oil caused a statistically significant increase in embryolethality at doses of 7.83 mg/kg per day and higher, with complete resorption in all dams at 31.33 mg/kg per day. When flubendazole was suspended in deionized water, a significant increase in embryolethality occurred only at a maternal dose of 125.32 mg/kg per day. The proportion of litters with anomalous fetuses was significantly increased at doses of 31.33 mg/kg per day and above when flubendazole was administered in deionized water, but increased at doses at four times lower when flubendazole was administered as in olive oil. Administered as a single dose in olive oil on any one of Days 6-12 of pregnancy, a flubendazole dose of 31.33 mg/kg caused significant increases in embryolethality and decreased fetal body weights on Days 7-9, with an 82.7% incidence of embryolethality on Day 8, with complete resorption in 5 of the 8 dams. The critical periods for teratogenic effects were between Days 8 and 11 of pregnancy, with Day 9 being the most critical. Fetuses with gross, skeletal, or internal anomalies were seen in dams given a single dose of as low as 7.83 mg/kg.     

Developmental toxicity of dibutyltin dichloride given on three consecutive days during organogenesis in cynomolgus monkeys

We previously reported that the administration of dibutyltin dichloride (DBTCl) by nasogastric intubation during the entire period of organogenesis, days 20–50 of pregnancy, was embryolethal, but not teratogenic, in cynomolgus monkeys. The present study was conducted to further evaluate the developmental toxicity of DBTCl given to pregnant monkeys on 3 consecutive days during organogenesis. Cynomolgus monkeys were given DBTCl at 7.5?mg/kg body weight/day by nasogastric intubation on days 19–21, 21–23, 24–26, 26–28, 29–31, 31–33, or 34–36 of pregnancy, and the pregnancy outcome was determined on day 100 of pregnancy. Embryonic/fetal loss was observed in 1 female given DBTCl on days 19–21, 2 females given DBTCl on days 24–26, and 1 female given DBTCl on days 34–36. There were no effects of DBTCl on developmental parameters in surviving fetuses, including fetal body weight, crown-rump length, tail length, or placental weight. No external, internal, or skeletal malformations were detected in fetuses in any group. DBTCl did not affect the incidence of fetuses with skeletal variation or skeletal ossification of fetuses. These data confirm our previous findings that DBTCl was embryolethal, but not teratogenic, in cynomolgus monkeys.     

LPS from Porphyromonas gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ET(B)) receptors in cultured endothelium-intact rat coronary arteries

The purpose of our study was to examine if lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g.) modifies the vasomotor responses to Endothelin-1 (ET-1) and Sarafotoxin 6c (S6c) in rat coronary arteries. The arteries were studied directly or following organ culture for 24 h in absence and presence of 2.5EU/ml LPS. The contractile responses of coronary arteries were investigated by using the selective ETB receptor agonist S6c (1 pM-0.3 μM) and ET-1 (1 pM-0.3 μM). The functional studies demonstrated an augmented contractile response only to S6c in isolated rat coronary arteries after organ culture (with or without LPS). These contractile responses by S6c were blocked by the selective ETB receptor antagonist BQ788 in both vessel groups. The augmented contractile response to S6c was supported by immunohistochemistry, where a significant increase in fluorescence intensity for ETB receptors in smooth muscle cells was observed after organ culture. The presence of LPS in the culture medium significantly increased the sensitivity of endothelium-intact coronary artery to S6c as compared to endothelium-denuded segments. Our results showed a significant increase in both ETB receptor protein levels and S6c-induced maximal contraction in coronary arteries upon 24 h of organ culture, which was further sensitized by LPS.     

In vitro embryotoxicity of petroleum creosote monitored via mouse preimplantation embryo culture.

A mouse preimplantation embryo culture system was utilized to characterize the in vitro embryotoxicity of petroleum creosote (PC), a complex mixture of aliphatic and polycyclic aromatic hydrocarbons. ICR mouse embryos, collected on d 3.5 of gestation (blastocyst stage), were exposed for 1 h to varying concentrations of petroleum creosote in serum-supplemented culture medium. Parallel embryo cultures were exposed to PC in medium supplemented with rodent hepatic S9 microsomal fractions to monitor the role of bioactivation in PC-induced embryotoxicity. Embryos were subsequently cultured in control medium for 72 h and observed for viability as well as specific, time-dependent developmental end points--hatching and attachment to the culture dish at 48 h, and trophoblastic outgrowth with a distinct inner cell mass at 72 h. Embryonic viability varied in inverse proportion to PC concentration. Petroleum creosote caused embryolethal effects at concentrations of 33 micrograms/ml of culture medium and 54 micrograms/ml. Embryotoxicity was not observed at 22 micrograms/ml. Culture supplementation with rodent hepatic S9 fractions did not modify, either qualitatively or quantitatively, the embryotoxicity of PC in vitro. These findings implicate PC as a prenatal toxicant and support environmental and human health concerns regarding PC exposure from PC-containing chemical waste sites.     

Effects of tartrazine on proliferation and genetic damage in human lymphocytes

The color additive, tartrazine (TRZ), is widely used in food products, drugs and cosmetics. Genotoxicity of TRZ and its metabolites has not been investigated in detail in the presence and absence of a metabolic activator (S9 mix) in human. Therefore, the aim of this study is to investigate the cytotoxic and genotoxic effects of TRZ and its metabolites on cultured human lymphocytes by using chromosome aberration (CA) and micronucleus (MN) tests. Cultures were treated with 625, 1250 and 2500?μg/ml of TRZ in the presence and absence of S9 mix. TRZ showed cytotoxic activity at the highest concentration due to significant decrease in mitotic index (MI) in the absence of S9 mix when compared with solvent control. TRZ and metabolites significantly increased the CAs and aberrant cells in the presence and absence of S9 mix at the higher concentrations. Increased MN values in cultures with and without S9 mix were found to significantly at the highest concentration when tested. Our results indicated that while both TRZ and its metabolites have genotoxic potential on human lymphocyte cultures with and without S9 mix, TRZ can induce cytotoxicity at the highest concentration in culture without S9 mix under the experimental conditions.     

Embryotoxicity of cisplatin in rats and mice

Cisplatin [cis-dichlorodiammineplatinum (II)], a broad spectrum antitumor agent, was tested for possible teratogenic and embryolethal effects on Wistar rats and Swiss Webster mice. Rats were given a single ip injection of 0.3, 1.0, 2.5, or 3.0 mg/kg cisplatin on Day 6, 8, 11, or 14 of gestation, whereas mice were given a single ip injection of 0.3, 3.0, 6.0, 8,0, or 13.0 mg/kg on Day 8 only. The embryonic LD50's in the rat were 2.88, 1.28, and 1.0 mg/kg for day 6, 8, and 11, respectively. There was no significant increase in embryolethality at any of the doses given on Day 14. The embryonic LD50 for mice was 5.24 mg/kg. An increase in the incidence of growth retardation or gross malformations was not discernable in the surviving fetuses with the number of dams used in this study. Cisplatin is highly embryolethal in rats and mice at dosages well below the adult therapeutic dosage in humans. This embryolethality is gestational stage-specific with the highest mortality corresponding to the period of rapid DNA replication in early organogenesis.     

Predicting the human teratogenic potential of the anticonvulsant, valproic acid, from a non-human primate model

The anticonvulsant, valproic acid (VPA) is a suspected human teratogen. This study, employing the rhesus monkey as an animal model, demonstrates that VPA has a significant teratogenic potential in the monkey. Timed pregnant monkeys were exposed orally to VPA at approx. 1X, 10X, and 30X (20, 200, and 600 mg/kg/day, respectively) the human therapeutic dose, daily, during organogenesis (gestation days 21-50). All fetuses of mothers exposed to greater than 1X exhibited some form of embryotoxicity. The highest dose, 30X, was 100% embryolethal, while offspring of the 10X dose group exhibited craniofacial and skeletal defects, and low body weights. Maternal pharmacokinetic parameters and plasma metabolites were determined for VPA on the first and last day of dosing for the 10X dose group. Comparison of the kinetic and metabolite data with that obtained for man indicates that the rhesus monkey is a good model for predicting the teratogenic potential of VPA in the human.     

Role of biotransformation in the in vitro preimplantation embryotoxicity of naphthalene

The in vitro developmental toxicity of the bicyclic aromatic hydrocarbon naphthalene was characterized with a preimplantation mouse embryo culture system. Day 3 ICR mouse blastocysts were co-cultured with naphthalene for 1 h either alone or in media supplemented with an Aroclor-induced rat S-9 preparation and cofactors. Toxin-treated blastocysts were subsequently cultured in NCTC 109 media with 10% fetal bovine serum for 72 h to observe the developmental effects of exposure. Developmental parameters observed included viability, hatching, culture dish attachment and trophoblastic outgrowth with the presence of a distinct inner cell mass. At media concentrations up to 0.78 mM, naphthalene alone exhibited negligible toxic effects in culture; however naphthalene co-cultured with Aroclor-induced rat hepatic S-9 fractions exhibited concentration-dependent embryolethality with an approximate LC50 of 0.18 mM in media. Naphthalene also induced concentration-dependent embryotoxicity at all observed parameters in S-9-supplemented media at concentrations ranging from 0.20 to 0.78 mM. These findings document the role of biotransformation in naphthalene's embryotoxicity to early mouse blastocysts and implicate naphthalene as a potentially embryotoxic and abortifacient component of polycyclic aromatic hydrocarbon mixtures.