Dimethyl Sulfoxide: Morphological, Histological, and Molecular View on Developing Chicken Liver

Abstract

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform's activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

Keywords: chicken embryo; cytochrome P450; development; dimethyl sulfoxide; liver; toxicity.

Figure 1

Relation between application dose of DMSO and mortality of chick embryos.

Figure 2

Relation between application dose of DMSO and body weight of chick embryo (CTR—control, * p < 0.01; ** p < 0.001; *** p < 0.0001).

Figure 3

Chicken embryo development on ED 9. Right position: physiological development, left position: general growth retardation with anophthalmia.

Figure 4

Malformations appearing after DMSO administration on ED 9. (A): Observation of local macroscopic colour changes on the liver after 10 μL of DMSO administration, scale bar 1 mm, (B): Haemorrhages on the limb buds after 15 μL of DMSO administration—arrows, scale bar: 1 mm, (C): Opening of the body wall, haemorrhages on the left-wing bud and pelvic region (arrows), malformation of the right limb bud—20 μL of DMSO administration, scale bar: 2 mm, (D): Growth retardation–25 μL of DMSO administration, scale bar: 2 mm.

Figure 5

(A): Microphotograph of the chicken liver in control group, H-E staining, scale bar: 50 μm, (B): Microphotograph of the chicken liver in experimental group—5 μL of DMSO administration, H-E staining, scale bar: 50 μm, (C): Microphotograph of the chicken liver in experimental group—10 μL of DMSO administration, black arrows—dilated bile canaliculi, H-E staining, scale bar: 20 μm.

Figure 6

(A): Microphotograph of the chicken liver in experimental group—25 μL of DMSO administration, scale bar: 10 μm, (B): Microphotograph of the chicken liver in experimental group—50 μL of DMSO administration, scale bar: 10 μm; S—liver sinusoid, black arrows—endothelial cells, white arrows—dilated bile canaliculi, Toluidine blue staining.

Figure 7

Transmission electron microscopy of the chicken developing liver. (A): Control group, magnification: 6500×; (B): Experimental group—25 μL of DMSO administration, magnification: 4000×; (C): Experimental group—25 μL of DMSO administration, magnification: 7500×; (D): Experimental group—25 μL of DMSO administration, magnification: 6800×; (E): Experimental group—50 μL of DMSO administration, magnification: 2800×; (F): Experimental group—50 μL of DMSO administration, magnification: 5100×; N—nucleus of hepatocyte, S—sinusoid, e—endothelial cell, Ec–erythrocyte, b—bile canaliculus, white arrow—lipid droplet, black arrows—intercellular junctions (A,F), black arrows—space of Disse (B,D).

Figure 8

Transmission electron microscopy of the chicken developing liver. (A): Experimental group—25 μL of DMSO administration, magnification: 8800×; (B): Experimental group—50 μL of DMSO administration, magnification: 4400×; arrows—slightly dilated mitochondria (A), fragmented cristae of mitochondria with electron-lucent matrix (B).

Figure 9

Relative gene expression from RT-qPCR analysis for genes CYP1A5, CYP3A37 and CYP3A80 (* statistically significant difference, p < 0.01; log-log transformed).