Alcohol exposure alters cell cycle and apoptotic events during early neurulation

Abstract

Background: Fetal alcohol exposure causes growth deficits, microencephaly, and neurological abnormalities. Although the effects of alcohol on developmental delay and growth-related deficits have been hypothesized, little is understood about how alcohol alters, in particular, the cyclin pathway within the cell cycle, which is critical to proliferation and apoptotic control. In this study, we examined cell cycle proteins pertinent to the G1-S phase transition and apoptosis, to determine if cell cycle misregulation can be attributed to apoptotic induction and growth defects.

Methods: We examined cell cycle regulation during G1 and S-phase, and DNA fragmentation damage, using E14 dorsal root ganglia neural stem cells (DRG-NC), and cultured mouse embryos exposed to 200 and 400 mg/dl ethanol.

Results: Alcohol-exposed DRG-NC demonstrated a dose-dependent increase in cells expressing increased cyclin D1 protein, and increased DNA fragmentation. Western blot analysis, using embryos, demonstrated an overexpression of cyclin D1, D2, and E2F1, key G1 to S-phase cell cycle regulatory components, and increases in p53, linking the cell cycle and apoptotic pathways. Bromodeoxyuridine incorporation indicated reduced DNA synthesis and growth in several embryonic regions. Propidium iodide staining demonstrated decreases in DNA content and increases in DNA fragmentation in several embryonic tissues.

Conclusions: This study indicated that retarded growth of DRG-NC and embryos, induced by alcohol, is associated with altered expression of cell cycle and apoptotic proteins and concurrent inhibition of proliferation and increased DNA fragmentation. We suggest that alcohol induces an increase in cyclin D1 expression, premature S-phase entry, and disjointed DNA synthesis with increased apoptosis.

Figure 4

Proliferation analysis by BrdU incorporation in dividing cells (controls, solid black; alcohol exposed, solid gray). (A) Alcohol reduces the amount of BrdU incorporation indicating S-phase cell cycle alterations during cell division. The reductions were evident in the neural tube, neural crest, lung buds, and atrium. (B) Mean density measures/equal area supports reduced proliferative cell counts from A. (C) Total area measures show marked reductions in neural crest cells, with increases in the surrounding head mesenchyme tissue (neural crest region), and the ventricle of the neural tube. In statistical analysis, Student's t-test was performed for P < 0.01, N = 4 independent animals for each group. Scale bars—A, B, D, E: 100 μm; C, D: 10 μm; E, F: 40 μm.

Figure 1

Alterations in DRG NSC growth and DNA fragmentation. DRG stem cells were exposed to 200 and 400 mg/dl alcohol and examined for DNA integrity and growth. (A) Alcohol concentration curve for DRG cell cultures over a 48 h period using 400 and 200 mg/dl. (B) Healthy control cells show compact DNA foci with PI staining (red). After alcohol exposure at either 200 or 400 mg/dl, cells show a considerable increase in DNA fragmentation (10 μm calibration). (C) Total cells in each culture were counted using Trypan exclusion, at 0, 6, 12, 24, and 48 h after alcohol treatment and are reported as a percentage of the starting cell count. A 40% decrease in cell numbers was seen within 12 h after alcohol treatment for either dose. (D) Cells with DNA fragmentation were counted using PI staining (red), and are reported as a percentage of total cell numbers. Significant damage was seen by 4 h. After 24 h of 400 mg/dl alcohol treatment, we found a >3.5-fold increase in apoptosis. ^*Denotes statistical significance between alcohol and control, ^** represents significant changes between control and other alcohol treatments. P < 0.05 Fisher's Student's t-test, N = 4.

Fig 2

Analysis of cyclin D1 expression and DNA integrity in DRG-NSC cultures. Cultured DRG-NSC (control or 400 mg/dl alcohol for 12 h) were labeled by immunohistochemistry for cyclin D1 (blue) then counterstained with propidium iodide (red). Low D1 expression was defined as a light and diffuse cytoplasmic blue fluorescence. High D1 expression was seen as bright blue focal fluorescence in both cytoplasmic and nuclear regions. Low PI staining was defined as pycnotic cells with little, compact DNA. High PI staining was represented by disrupted DNA foci with >2-fold normal DNA content. Cells typical of control cultures are seen in (A, B). (A) Shows high D1 with low PI content representative of late G1 phase (arrowhead), or high PI with low D1 (arrow) representing S/G2 phase. (B) Shows low PI with low D1 label representative of mitosis (arrows and B arrowhead). Many cells have an increase in DNA fragmentation (C and D arrows) with an accompanying high cyclin D1 expression (arrowhead) after alcohol exposure, suggesting an apoptotic cell population with a high G1 marker. Labeled cells from control cultures, or those exposed to 200 or 400 mg/dl for 12 h, were counted for either high or low expression of cyclin D1 and DNA content. Graph demonstrates a dose-dependent alcohol-induced increase in cyclin D1 with 400 mg/dl alcohol, suggesting an increased population of cells in G1 or early S-phase. In statistical analyses, Fisher's Student's t-test was performed for N = 3. ^* Denotes significance between control and alcohol treatment. ^** Denotes significance between control and alcohol groups, P < 0.03. Scale (A, C) 10 μm; (B, D) 5 μm.

Figure 3

Western blot analysis of embryonic cell cycle proteins. Both alcohol and control protein (40 μg) samples were run on SDS–PAGE gels and detected by chemical luminescence. GAPDH was used as an internal control. Statistical analysis of band density by Sstudent's t-test showed that alcohol increases protein expression of E2F1, cyclin D1, cyclin D2, p53, and phosphorylated p53 (P ≤ 0.005).

Figure 5

Comparison of BrdU incorporation for regions of the developing embryo. Controls (A, C, E, G) versus alcohol-treated (B, D, F, H). Alcohol induced decreases in BrdU incorporation in the neural tube (A, B, C, D), heart atrium and ventricle (G, H), and lung bud and trachea (E, F). Alcohol also induced increases in the size of the heart ventricle (G, H), and neural tube ventricle. ^*Denotes the neural tube ventricle in (A), and altered structural integrity of the mesenchyme of the neural crest including neural crest cell populations (A, B labeled). Arrows depict regions of distinct change in BrdU incorporation. Distinct alterations in BrdU-labeled cells were seen in the neural tube, with alcohol-induced proliferation of cells away from the ventricle walls, often in clusters (D arrow heads). V, neural tube ventricle. Scale markers—A, B, G, H: 100 μm; E, F: 40 μm; C, D: 10 μm.

Figure 6

Fluorescent micrographs for cyclin D1 (green) and propidium iodide (red) from areas of control and alcohol exposed E8.25 + 2 embryos. Arrows depict regions with distinct alterations in DNA content, and correlations to altered cyclin D1. In the limb buds, alcohol-induced decreases in overall cyclinD1 and DNA, with increases in developing vasculature (A, 1, 1′). In the caudal regions of the neural tube, alcohol-induced overlapping increases in cyclin D1 and DNA content in the floor plate (2′), areas of developing somite mesenchyme (2) and hematopoietic cells ventral to the notochord (2″). A distinct region in the neural tube roof plate shows a cell population with high D1 staining, suggesting a synchronized proliferation (B). In the developing heart overall decreases are seen in D1 and DNA. However, several overlapping increases in both D1 and DNA are seen in the atria (3,3″) and ventricular walls (3′). Areas of the rostral neural tube, including the floor plate and lateral walls, show an overlapping increase in expression of both markers (4″, 4, and 4′, respectively) from alcohol. DNA is increased throughout the neural tube, with no significant change in overall D1. Scale bars: limb buds 25 μm, all others 100 μm.

Figure 7

Histograms for fluorescent intensity measurements from the heart and neural tube for both cyclin D1 and propidium iodide. ^*Denotes those giving statistically significant values in a Fisher's Student's t-test (P < 0.03). Three serial sections were used from three independent animals.

Figure 8

Fluorescent microscopy of propidium iodide staining in the embryo heart and neural tube. (A) PI staining in control neural tube showing proliferating cells lining the neural tube along the ventricle. (B) Alterations in DNA content after alcohol exposure in the neural tube; note changes in overall DNA content with increases in cells of the neural tube away from the ventricle and the floor plate. (C) PI staining in the heart for control animals. (C′) PI staining in the developing septum of control animals. (D) PI staining in the alcohol-exposed embryo shows distinct changes in the septum, cardiac gel, and walls of the ventricle and atrium. (D′, D″) Extensive DNA fragmentation is seen in many areas of the embryo heart after alcohol exposure. Scale bars neural tube 25 μm, heart 10 μm and, lower panel, heart 100 μm. V, neural tube ventricle; FP, neural tube floor plate; A, heart atrium; S, atrial/ventricular septum; HV, primitive heart ventricle.