Evaluation of teratogenicity and genotoxicity induced by kramecyne (KACY)

Abstract

Kramecyne (KACY), a polymer isolated from Krameria cytisoides Cav, has anti-inflammatory, anti-nociceptive, anti-arthritic and anti-ulcerogenic properties. As a part of standard preclinical safety tests, the present study sought to determine potential developmental toxicity (in female rats) and genotoxicity (in male mice) of KACY. Pregnant female rats were divided into six groups: the negative control (vehicle), the positive control (250 mg/kg of acetylsalicylic acid (ASA)), and four experimental groups (50, 250, 500 and 1000 mg/kg of KACY). To evaluate genotoxicity by in vivo micronuclei (MN) and sister chromatid exchange (SCE) tests, male mice were divided into five groups: the negative control (vehicle), the positive control (1.5 and 2.5 mg/kg of doxorubicin for MN and SCE, respectively), and three experimental groups (50, 500 and 1000 mg/kg of KACY). All treatments were administered by oral gavage. A slight maternal toxicity was evidenced by lower weight gain for rats receiving 500 and 1000 mg/kg of KACY, but no fetal malformations were found. However, there were less live fetuses/litter and greater post-implantation loss/litter at these two doses. Manifestations of developmental toxicity were limited to a higher rate of skeletal alterations. The MN tests did not evidence genotoxicity or cytotoxicity. KACY caused a slightly but significantly increased frequency of SCE. Although KACY-treated rats had skeletal alterations, these apparently were not caused by a mechanism of genotoxicity. Furthermore, the same administration in adult male mice did not produce genotoxicity. Hence, KACY herein proved to be safe for rats during the period of organogenesis.

Keywords: Kramecyne; Micronuclei; Sister chromatid exchange; Teratogenic study.

Fig. 1

Structural formula of kramecyne; a cyclic hexamer polymer (A) and a monomer (B).

Fig. 2

External anomalies: (A) a normal fetus of the negative control; (B) a fetus from a female rat administered KACY at 1000 mg/kg shows small size; (C–E) fetuses treated with ASA at 250 mg/kg show exencephaly (C), craniorachischisis (D), and gastroschisis and exencephaly (E).

Fig. 3

Transverse sections of brain and kidneys of rat fetuses. The brain (A) and kidney (D) sections of control rat fetuses. The brain (B) and kidney (E) sections of fetuses from female rats treated with ASA (250 mg/kg). Dilation of the lateral and central brain ventricles can be appreciated, as well as dilation of the renal pelvis. The brain (C) and kidney (F) sections of fetuses from females treated with KACY (1000 mg/kg). A slight dilation of the renal pelvis is noticeable.

Fig. 4

Fetal skeletal anomalies: A and D, a fetus of the negative control group. B and E, a fetus showing acrania (dashed line), extracted from a female rat treated with 250 mg/kg of ASA. C and F, a fetus showing poor ossification of frontal, parietal and supraoccipital bones (arrows heads), extracted from a female rat treated with 1000 mg/kg of KACY.

Fig. 5

Fetal skeletal anomalies in sternebrae (A–C) and ribs (D–F). Fetus of the negative control (A, D); fetus from a female rat treated with 250 mg/kg of ASA, showing poor ossification in sternebrae (B, arrows heads) as well as wavy ribs, lobed ribs and poorly ossified vertebrae (E, arrows heads); fetus from a female rat treated with 1000 mg/kg of KACY, showing poor ossification in sternebrae (C, arrows heads) as well as rudimentary ribs (F, arrows heads).

Fig. 6

Number of micronucleated polychromatic erythrocytes (MNPE) found in the peripheral blood of control, DXO-treated and KACY-treated mice. Each bar represents the mean ± SEM of MNPE in 1000 polychromatic erythrocytes per mouse (n = 6). DXO = doxorubicin; KACY = kramecyne. ^aA significant difference (p < 0.001) in relation to the control.

Fig. 7

A mouse peripheral blood smear showing a polychromatic erythrocyte with a micronucleus.

Fig. 8

Number of polychromatic erythrocytes in mouse peripheral blood from the control, DXO-treated and KACY-treated groups. Each bar depicts the mean ± SEM found in 1000 polychromatic erythrocytes per mouse (n = 6). DXO = doxorubicin; KACY = kramecyne. ^aA significant difference (p < 0.001) in relation to the control.

Fig. 9

Sister chromatid exchange (SCE) found in the bone marrow cells of control, DXO-treated and KACY-treated mice. Each bar represents the mean ± SEM detected in 30 s-division metaphases (n = 6). DXO = doxorubicin; KACY = kramecyne. A significant difference (p < 0.05) in relation to the ^acontrol and/or ^bDXO at 2.5 mg/kg.

Fig. 10

Cumulative frequency of sister chromatid exchanges (SCEs) per bone marrow cell of control, DXO-treated and KACY-treated mice. Each line portrays the cumulative frequency of SCE displayed in 30 s-division metaphases per group (n = 6). DXO = doxorubicin; KACY = kramecyne.

Fig. 11

A second-division bone marrow mitosis showing sister chromatid differentiation: a single sister-chromatid exchange (A), and eight sister-chromatid exchanges (B).