Cell experiment:

In a 3-cm dish, 4 × 104 cells are seeded in 2 mL of medium. The next day, Ifosfamide is added to final concentrations from 0 to 5 mM. After six additional days the medium is removed, the cells washed with PBS, and either stained or counted[2].

Animal experiment:

Rats[1]Prior to mating, female rats are divided into four groups of eight: group 1, untreated negative controls; group 2, rats injected i.p. with 1 mL 0.9% NaCl; group 3, rats injected i.p. with 25 mg/kg Ifosfamide; group 4, rats injected i.p. with 50 mg/kg Ifosfamide. After injection of Ifosfamide once daily for 5 consecutive days, three females are placed in a cage with one untreated male for up to 1 week. Vaginal smears are examined daily to determine pregnancy. The first 24 h period following mating is designated day one of pregnancy if sperm are detected. The pregnant females are housed singly and observed daily for signs of toxicity and abortion. All pregnant animals are sacrificed by decapitation on day 18 of gestation. Cardiac blood (2.5 -3 mL/rat) is collected in nonheparinized test tubes, centrifuged at 3,000× g for 30 min and the serum is decanted and stored at -70℃ until used for hormone assay. After blood collection, uteri and both ovaries are removed, washed in saline solution and the corpora lutea of pregnancy are counted visually, and the number of implantation sites, resorption sites and viable fetuses are counted in each uterine horn. Each fetus is removed from its umbilical cord, weighed and the crown rump (CR) length is measured. Fetuses are examined for external malformation and the placental weights are recorded. Fetuses and placentas of control and treated groups are fixed in 10% neutral buffered formalin for immunohistochemical and histological examination[1].

Ifosfamide is an alkylating chemotherapeutic agent with activity against a wide range of tumors.

Ifosfamide is an alkylating chemotherapeutic agent with activity against a wide range of tumors[1]. Ifosfamide is activated by the cytochrome P450 family. Ifosfamide (0-5 mM) suppresses the viability of CYP2B1-expressing C8III-1 cells, while it is cytotoxic to the non-CYP2B1-expressing CrFK cells only at high concentration (5 mM)[2]. CYP BM3 mutants activates Ifosfamide, and Ifosfamide shows inhibitory activity against human U2OS cells[3].

Ifosfamide (50 mg/kg, i.p.) treatment before mating increases percentage of post-implantation loss and resorbs fetuses in pregnant rats. Ifosfamide also (50 mg/kg, i.p.) decreases the progesterone in the serum of pregnant rats. However, Ifosfamide causes no obvious difference with the control rats at 25 mg/kg. Ifosfamide (25, 50 mg/kg, i.p.) induces apoptosis and histological changes in the placentas and prenatal rats, most sensitive fetal organs are the brain, liver and kidney[1].

References:
[1]. Helal M. Prenatal effects of transplacental exposure to ifosfamide in rats. Biotech Histochem. 2016 Jul;91(5):357-68.
[2]. Karle P, et al. Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide. Cancer Gene Ther. 2001 Mar;8(3):220-30.
[3]. Vredenburg G, et al. Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants. Toxicol Lett. 2015 Jan 5;232(1):182-92.