Studying significance of apoptosis in mediating tolbutamide‐induced teratogenesis in vitro

Abstract

The incidence of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus) is growing worldwide and poses a serious public health problem in a current paradigm of changing life style and food habits. Tolbutamide (sulfonylurea) is among the commonly used anti-diabetic drugs worldwide for treating type 2 diabetes and is known to cause congenital malformations in animals. In this study, the effect of tolbutamide on major organogenesis period and the possible involvement of apoptosis in mediating congenital malformations have been carried out. In the present study design, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of tolbutamide, i.e., 10, 100, and 1000 μg/mL cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The tolbutamide decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to tolbutamide at 10 μg/mL culture did not show any significant effect on embryonic growth and development in vitro. In parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3'-OH labeling of cultured rat embryos to examine the role of apoptosis in bringing about tolbutamide-induced teratogenesis. All results were found to be dose dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The outcome of the research suggested that apoptosis might be involved in mediating teratogenesis of tolbutamide in vitro. Further research is warranted to fully understand this mechanism.

Investigation of Developmental Toxicity and Teratogenicity of Antiemetics on Rat Embryos CulturedIn Vitro

“…With this model, rat embryos were cultured in vitro from gestational day 9.5 to 11.5, which is the critical period of organogenesis in the rat, equivalent to 3–6 weeks after fertilization in human embryos. This method has been used extensively in studies in the field of teratogenesis and toxicology (Webster et al., ; Hewitt et al., ; Karabulut et al., , ; Uysal et al., ; Longo et al., ; Singh and Sinha, ; Zhang et al., ; Singh et al., ).…”

Role of apoptosis in mediating diclofenac-induced teratogenesis

“…However, their number at LD (3.75 mg/ml)-culture was not found to be significant with respect to untreated (control; p > 0.05). As discussed in earlier studies, a sub-G0 peak could be another qualitative parameter of apoptosis (Darzynkiewicz et al, 1997;Del Bino et al, 1999;Singh and Sinha, 2010;Singh et al, 2005Singh et al, , 2009Singh et al, , 2011. Hence, the sub-G0 peak was employed as an additional confirmatory indicator of apoptosis.…”

“…The supernatant was discarded and 0.5 ml of the binding buffer was added to the tubes and 10 ml of propidium iodide (PI; stock solution, 50 mg/ml) was later added and cells were then analyzed using fluorescenceactivated cell sorter (FACS) caliber (all the chemicals and reagents were supplied in kits). Flow cytometry analysis was performed using a single laser-emitting excitation light beam at 488 nm that enabled the detection and quantitation of embryonal cells as described previously (Singh et al, 2011). In brief, cells were sorted into three fractions: (1) viable cells (annexin V negative and PI negative), (2) apoptotic cells (annexin V positive and PI negative) and (3) necrotic/late apoptotic cells (annexin V positive and PI positive), respectively.…”

50 Years Ago in

“…There is also experimental evidence that tolbutamide exposure to developing rat embryos increases apoptosis-mediated markers, including annexin V binding and DNA fragmentation, in a dose-dependent fashion. 1 Although apoptosis is a necessary mechanism for normal embryologic development, based on this experimental evidence, it is hypothesized that tolbutamide-mediated teratogenesis occurs through dysregulated apoptosis.…”